Protein complex having activity catalyzing asymmetric oxidation reaction and process for producing the same

ABSTRACT

A process for producing a cross-linked crystallized protein complex, which comprises: a first step of concentrating a crude protein derived from an animal or plant; a second step of encapsulating the protein in a gel, to thereby allow the protein to undergo air oxidation, and then extracting a protein complex from the gel; a third step of allowing the extracted protein complex to undergo crystallization and precipitation; and a fourth step of cross-linking the precipitated protein complex. Alternatively, by use of a fifth step of drying (FD) the obtained crosslinked crystallized protein complex, to thereby form a powder. As a result, there is provided an enzyme which is stable at room temperature storage, and has an activity in catalyzing an asymmetric oxidation reaction. That is, there is provided a useful material which enables an efficient enzyme-mimetic reaction under a mild condition.

TECHNICAL FIELD

The present invention relates to a protein complex which at leastcomprises a protein and calcium, and also has an activity in catalyzingan asymmetric oxidation reaction, and a process for producing the same.The protein complex can be obtained, for example, by encapsulating (orincluding) a water-soluble crude protein which can easily be separatedfrom an animal or plant tissue, in a gel (calcium alginate gel, etc.),so as to allow the protein to undergo air oxidation, and then separatingthe protein complex from the resultant oxidation product.

BACKGROUND ART

In various fields including the fields of pharmaceuticals and foods,there has been increasing necessity of a technique of separating anoptical isomer to thereby collect the one useful optical isomer. Forexample, there has been studied a method wherein drugs and perfumes areproduced by using an immobilized enzyme having a function of selectivelyoxidizing/reducing or deracemizing one of optical isomers.

A chemical change in vivo is usually catalyzed by an enzyme. Abioreactor is exemplified as one of the systems of utilizing such anaction or mechanism of these enzymes in the production or synthesis ofuseful substances.

An immobilized enzyme, which has been bonded to an insoluble carrier,takes a leading part of the bioreactor. By use of the immobilizedenzyme, a product can easily be separated from the enzyme serving as acatalyst. The immobilized enzyme has widely been used in the fields ofresearch, medical care, analysis and industry. The central part of thebioreactor causing a chemical reaction is a reaction element (or areaction device), and a purified enzyme, organelle or cell per se isused for the purpose of converting a raw material into a product, or ofanalyzing by utilizing a chemical change. Since the reaction elementmust remain in a rector and can be repeatedly used, the reaction elementis immobilized by various methods (with respect to details ofutilization of these enzymes, for example, it is possible to refer to“Kagaku Zokan (i.e., Chemistry, Special Issue) No. 119, Production ofUseful Substances by Hybrid Process”, Kagaku Dojin; “Bioreactor” editedby Saburo Fukui, Biotechnology Series, Kodansha Ltd., “ImmobilizedEnzyme”, edited by Ichiro Chibata, Kodansha Scientific Ltd.).

However, since the enzyme requires much cost for purification and thepurified enzyme is often unstable, it leaves room for improving the costburden for stabilization thereof. For this reason, there is an examplewherein microorganism containing the objective enzyme was immobilized asit is, in place of the purified enzyme. Examples thereof may include anexample wherein microorganisms including aspartase are immobilized, tothereby produce L-aspartic acid, and an example wherein L-alanine iscontinuously produced by using aspartic acid to be produced in thisplant as a raw material (see “Kagaku Zokan No. 119, Production of UsefulSubstances by Hybrid Process”, Kagaku Dojin; “Bioreactor” edited bySaburo Fukui, Biotechnologies Series, Kodansha, Ltd.; “ImmobilizedEnzyme”, edited by Ichirou Chibata, Kodansha Scientific Ltd. and thelike).

With respect to a redox catalyst seen from an industrial point of view,e.g., in “Chemical and Industry (i.e. Kagaku To Kogyo)”, Vol. 62-1,January 2009, pp. 44-45 (through the development of a biocatalyst), itis considered that a method of utilizing functions of dehydrogenase andcoenzyme, which are present in cells, such as microorganisms, yeasts andcultured plant cells, as they are, is dominant in view of cost, and thatextra cost burden required to isolate and purify oxidoreductase andcoenzyme from biont is not worth the costs of an operation ofstabilizing an enzyme, and conjugating a reaction (ketone→alcohol) witha reaction (coenzyme NADH→NAD⁺) or a reverse reaction thereof.

In recent study of catalyst design to be replaced by the enzyme, thereis proposed an example wherein an enzyme-like active domain was producedby introducing a metal complex into a “crude protein”.

For example, as known in “Protein, Nucleic Acid, Enzyme”, 2004,November, Vol. 49, No. 14—Molecular Design of Metalloenzyme, Mainly HemeEnzyme—(Graduate School of Science, Nagoya University; YoshihitoWatanabe), it is disclosed that oxidation activity occurs even if hem(iron) in an active domain of chloroperoxidase (CPO) is replaced byanother metal complex, and it is disclosed that design of appropriatearrangement of a functional amino acid residue in the vicinity of themetal to be arranged is important for the construction of an activedomain.

According to the document “Tetrahedron Letters” No. 44, pp. 4281-4284(1978) (Asymmetric Reduction of aryl trifluoromethyl ketones with anmodel compound in a chiral hydrophobic binding site of sodium cholatemicelle, β-cyclodextrin and bovine serum albumin) “Naomichi Baba et al.;Institute for Chemical Research, Kyoto University”, asymmetric reductionis carried out by reacting substrate trifluoromethyl-acetophenone in thepresence of 1-propyl-1,4-dihydronicotinamide (NAH) or sodium borohydride(NaBH₄) using, as the catalyst, components other than enzyme:surfactant-like bile acid (NAC), β-cyclodextrin (β-CD) and bovine serumalbumin (BSA). The above results reveal that an active domain (stericconfiguration R, optical purity of 46.6% ee) is also present in bovineserum albumin (BSA).

Japanese Patent No. 3,294,860 (a process for producing an opticallyactive alcohol) discloses an example wherein an optically active alcoholwas resolved with an optical purity of about 100% ee from a crudeprotein derived from animals and plants, and an optically active alcohol(100% ee, yield of 50%) is synthesized by using the first step ofextracting a water-soluble protein from grains or beans; second step ofencapsulating the protein in a calcium alginate gel; and third step ofcarrying out an asymmetric oxidation conversion reaction of substrateusing the encapsulated protein as a catalyst in combination. In JapanesePatent No. 3,683,129 (a process for producing an optically activealcohol), an optically active alcohol (100% ee) is synthesized by usingthe first step of extracting a water-soluble protein selected from eggwhite and ovalbumin separated from egg white; the second step ofencapsulating the protein in calcium alginate; and the third step ofcarrying out an asymmetric oxidation conversion reaction of thesubstrate using the encapsulated protein as a catalyst in combination.

In general, the method of producing an immobilized enzyme is typicallythe follow methods:

(1) a carrier binding method wherein the extracted and purified enzymeis bound to a water-insoluble carrier, for example, derivatives ofpolysaccharides, such as cellulose, dextran and agarose; apolyacrylamide gel and the like;

(2) a cross-linking method wherein the extracted and purified enzyme isimmobilized by forming a cross-link between he extracted and purifiedenzymes using a reagent having two or more functional groups; and

(3) a (micropcasule type) encapsulating method wherein the extracted andpurified enzyme is incorporated in a fine matrix of a gel, for example,a gel such as alginate, starch, konjak (devil's tongue jelly),polyacrylamide gel or polyvinyl alcohol (matrix type) or coated with asemitransparent membrane.

A cross-linked enzyme crystal (CLEC) method which appeared in the 1990sis a method wherein the extracted and purified enzyme is crystallizedusing ammonium sulfate, polyethylene glycol (PEG) and the like and thencross-linked using a polyhydric modification reagent such asglutaraldehyde (GA), and is used most practically as an industrialimmobilized enzyme technique. With respect to the CLEC method, forexample, ChiroCLEC (enzyme for the synthesis of chiral compounds) ismade into a product as an enzyme for organic synthesis by Altus Co. Withrespect to ChiroCLEC-BL, subtilisin derived from Bacillus licheniformisis immobilized and then cross-linked and solid-phased. With respect toChiroCLEC-CR, lipase derived from Candida rugosa is immobilized and thencross-linked and formed into a solid-phase.

These products exhibit stably activity even in an organic solvent andare also excellent in thermostability. They have a feature thathydrolysis or acylation of carboxylic acid, alcohol, amino acid, esterand the like can be carried out while maintaining optical activity. Thecross-linked enzyme crystal (CLEC) (1) can optimize a function of anenzyme under operation conditions that the enzyme is cross-linked andimmobilized, (2) can be developed in various commercially availableenzymes such as hydrolase, oxidoreductase and lyase, and (3) can bedeveloped by an enzyme capable of producing transgenic microorganismmodified so as to meet specific needs.

Japanese Examined Patent Publication (JP-B; KOKOKU) No. 68914 (a processfor immobilizing an enzyme) discloses an example wherein an enzyme isadsorbed to an aminated silica gel of a porous water-insoluble carrier,and then the enzyme is immobilized by a covalent binding reaction usinga polyfunctional cross-linking agent (glutaraldehyde). In this case,drawbacks of the enzyme, which is likely to leave from a carrier becauseof a weak binding force between a carrier and an enzyme, is solved bycross-linking the enzyme to a polyfunctional cross-linking agent. Aremarkable improvement in half life of activity of an immobilizationcarrier has been realized by using, in addition to enzyme adsorptivityof porous carriers such as a porous aminated silica gel and a porousaminated zeolite, a polyfunctional cross-linking agent (glutaraldehyde).

Known advantages of the cross-linked enzyme crystal (CLEC) aresummarized as follows. Crystallization of the enzyme means that watermolecules coated around enzyme molecules are removed by the addition ofammonium sulfate and polyethylene glycol and thus enzyme molecules beginto be polymerized with each other, and means that molecules finallybecomes large, resulting in the precipitation thereof. The meaning ofthe crystallization is different from that in the case of an organiccompound wherein a solution of the organic compound is cooled, tothereby solidify the compound.

(Advantages of Cross-Linked Enzyme Crystal)

-   -   High-purity enzyme is not required (applicable to a partially        purified prepared product)    -   Simple operation and wide application    -   Stable at room temperature for a long period (one or more years)    -   Substantially 100% active protein (high-volume measurement        (volumetric) and catalyst productivity)    -   Easy recovery and recycling    -   High-temperature stability and resistance to organic solvent as        compared with enzyme alone    -   High activity and selectivity (may be sometimes higher than        those of enzyme alone)    -   There's no need to filtrate an enzyme in an aqueous medium    -   Quick optimization (using HTE) shortens a development time    -   Combi CLEA containing one or more enzymes for catalytic cascade        process

Examples of market and possibility of application of the cross-linkedenzyme crystal (CLEC) include CLEC synthesis (drugs, perfumes and tastesubstances, pesticides, functional foods, fine chemicals, bulkymonomers), foods and beverages, pulps and papers, cosmetics, oils andlipids, woven fabrics, waste treatment, surfactants, biosensors,diagnostic drugs, protein transport and the like.

With respect to dehyrdrogenases, microorganisms-derived alcoholdehydrogenase (ADH) from Rhodococcus eryth ropolis, and formatedehydrogenase (FDH) from Candida boidinii are known as enzymes forpreparation of CLEA, at present.

PRIOR ART DOCUMENTS Patent Document

-   Patent Document 1: Japanese Patent No. 3,294,860-   Patent Document 2: Japanese Patent No. 3,683,129-   Patent Document 3: JP-B No. 4-68914

Non-Patent Document

-   Non-Patent Document 1: “Kagaku Zokan No. 119, Production of Useful    Substances by Hybrid Process”, Kagaku Dojin, “Bioreactor” edited by    Saburo Fukui-   Non-Patent Document 2: Biotechnologies Series, Kodansha Ltd.,    “Immobilized Enzyme”, edited by Ichirou Chibata, Kodansha Scientific    Ltd.-   Non-Patent Document 3: “Chemistry and Industry (i.e., Kagaku To    Kogyo), Vol. 62-1, January 2009, pp. 44-45-   Non-Patent Document 4: “Protein, Nucleic acid, Enzyme” 2004,    November, Vol. 49, No. 14, —Molecular Design of Metalloenzyme,    Mainly Heme Enzyme—(Graduate School of Science, Nagoya University;    Yoshihito Watanabe)-   Non-Patent Document 5: “Tetrahedron Letters” No. 44, pp.    4281-4284 (1978) (AsymMetric Reduction of aryl trifluoromethyl    ketones with an model compound in a chiral hydrophobic binding site    of sodium cholate micelle, β-cyclodextrin and bovine serum albumin)    “Naomichi Baba et al.; Institute for Chemical Research, Kyoto    University”-   Non-Patent Document 6: “Food and Technology (i.e., Shokuhin To    Gijutshu)”, October, pp. 1-9 (2008) (Role of Table or Common Salt in    Network Formation of Gluten Protein), “Reiko Urade; Graduate School    of Agriculture, Kyoto University”-   Non-Patent Document 7: Crystal structure of the DsbB-DsbA complex    reveals a disulfide bond severation mechanism-Cell 127,    789-801 (2006) (Kenji Inaba et al.; Medical Institute of    Bioregulation, Kyushu University)-   Non-Patent Document 8: —STRUCTURE-FUNCTION STUDIES OF GLUTAREDOXINS    AND RELATED OXIDOREDUCTASRE—” (Tobias H. ElGAn; From THE DEPARTMENT    OF BIOSCIENCES AND NUTRITION Karolinska Institutet, Stockholm,    Sweden)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to overcome the above-mentioneddrawback of the prior art, and to provide a useful material whichenables an efficient enzyme-mimetic reaction under a mild condition.

Another object of the present invention is to provide a material havingan “enzyme-mimetic” reaction activity, which is excellent inenvironmental aspects and safety.

Still another object of the present invention is to provide a suitableprocess for producing a material having such “enzyme-mimetic” reactionactivity.

Means for Solving the Problems

As a result of an earnest study, the present inventors have found aprotein complex having an activity in catalyzing an asymmetric oxidationreaction.

The protein complex of the present invention has been made based on theabove discovery, and is more specifically a protein complex whichcomprises at least protein and calcium, and also has an activity incatalyzing an asymmetric oxidation reaction.

The present inventors have further made a progress of a study based onthe above discovery and found a method capable of efficiently obtainingthe above protein complex having useful properties. The productionmethod of the present invention has been made based on the abovediscovery, and more specifically includes the first step ofconcentrating a crude protein from a water-soluble moiety derived fromanimal and plant; the second step of encapsulating the protein in a gel,to thereby allow the protein to undergo air oxidation, and thenextracting a protein complex from the gel; the third step of allowingthe extracted protein complex to undergo crystallization precipitationin an aqueous solution; and the fourth step of cross-linking theprecipitated protein complex.

According to an aspect of the above production method, it is possible toinclude the first step of concentrating a crude protein from awater-soluble moiety of animal and plant; the second step ofencapsulating the crude protein in a calcium alginate gel, exposing thegel to air for several times (air oxidation), shaking in warm water andthen extracting a protein complex; the third step of subjecting theprotein complex to saturated 30% ammonium sulfate precipitate; and thefourth step of cross-linking the precipitated crystallized proteincomplex.

According to another aspect of the present invention, an inexpensivecrude protein derived from animal and plant is encapsulated in a calciumalginate gel and the gel beads (in the presence of oxygen and dissolvedcalcium) are subjected to air oxidation, to thereby induce (i)intermolecular disulfide bond, (ii) intermolecular aggregation (<6.4 Å)and (iii) change in water solubility (protein complex formation), andthen the complex can be suitably extracted by shaking in warm water.

According to an aspect of the present invention, it is also possible tosynthesize an optically active alcohol of about 100% e.e. by asymmetricoxidation of one enantiomer of a substrate racemic alcohol even in caseof using a protein complex or a cross-linked crystallized proteincomplex, and also to provide a specific production method.

According to an aspect of the present invention, it is also providedthat a protein complex of a crude protein derived from animal and planthas, in addition to a reaction of selective asymmetric oxidation to oneenantiomer of a secondary alcohol substrate having a benzene skeleton(or structure) or a naphthalene skeleton (yield of 50%), deracemizationreaction activity (yield>85%) of alkyl-chain secondary alcohols havingno skeleton mentioned above, such as Matsutakeol.

The subject matter of present invention is not the production of theabove-mentioned purified enzyme derived from microorganisms, transgenicenzyme derived from microorganisms, or microorganisms or transgenicmicroorganisms, and immobilized enzyme derived from animal tissue(liver, pancreas, etc.) and immobilized microorganism, and it is one offeatures of the present invention that the subject matter thereof is amore inexpensive crude protein derived from animal and plant resourceswhich does not require the step of isolation and purification of anenzyme at high cost. Examples of the “plant resource” include grainssuch as buckwheat, amaranth, rice, wheat, barley, corn, oats, rye,foxtail millet, barnyard millet, millet, adlay and sorghum; beans suchas adsuki beans, kidney beans, green peas, green beans and soy beans;and the respective plant tissue of seeds, leaves, stems, roots, flowersand fruits of general grasses and weeds further included therein.

Examples of the “animal resource” include those derived from chicken eggas egg white or ovalbumin, and egg albumin of chickens, amphibians andfishes can be similarly used. There is no limitation on those derivedfrom chicken egg, and protein origin is not limited to egg.

In an aspect of the present invention, as mentioned below, it alsobecomes possible to produce a protein complex, which is environmentallyfriendly and is low-cost and also has high storability, wherein thefirst step of extracting a water-soluble protein from grains, beans, andthe respective plant tissue of seeds, leaves, stems, roots, flowers andfruits of general grasses and weeds further included therein; the secondstep of encapsulating the protein in a calcium alginate gel, to therebyallow the protein to undergo air oxidation, shaking in warm water at 40°C. for 10 hours or more, and separating an aqueous protein complexsolution fraction from the beads; the third step of adjusting theaqueous protein complex solution fraction to 30% saturated ammoniumsulfate, to thereby form a precipitate; the fourth step of cross-linkingthe obtained crystallized protein complex using glutaraldehyde; and thefifth step of drying (FD) the obtained crosslinked crystallized proteincomplex, to thereby form a powder in combination.

Effects of the Invention

As described above, according to the present invention, there is/areprovided a protein complex derived from plant and/or a protein complexderived from animal, imparted with a practical asymmetric oxidationactivity. According to the present invention, there is further provideda process for producing a protein complex, which is suited for thecross-linked crystallized protein derived from animal and plant, and isenvironmentally friendly and realize low cost production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a persistence ratio of a substrate whendistilled water is added to a green pea protein-calcium alginate gelbeads, and a substrate R-1-(2-naphthyl)ethanol is added at apredetermined time after initiation of shaking in the second step of theproduction method of the present invention.

FIG. 2 a is a graph wherein a ratio (%) of a substrate to a product witha reaction time is monitored when various substrates are added at 10hours after blank shaking in the second step of the production method ofthe present invention.

FIG. 2 b is a graph wherein a substrate R-1-(2-naphthyl)ethanol is addedto an aqueous solution or the like at 10 hours after blank shaking andthen an amount (%) of the formed ketone is monitored in the second stepof the production method of the present invention.

FIG. 2 c is a graph wherein green pea-calcium alginate gel beadsprepared by added dropwise in a different concentration of calciumchloride is blank-shaken for 10 hours and then reacted with a substrateR-1-(2-naphthyl)ethanol, and a protein complex activity is examined inthe second step of the production method of the present invention.

FIG. 2 d is a graph wherein green pea-calcium alginate gel beads areprepared in a predetermined mass of a green pea protein and blank-shakenfor 10 hours and then and a substrate R-1-(2-naphthyl)ethanol (50 mg) isrespectively added and a protein complex activity is examined in thesecond step of the production method of the present invention.

FIG. 3 shows determination of a molecule weight of water-soluble proteincomponent (SDS-page) obtained by shaking a green pea-Ca alginic acid gelin warm water for 10 hours or more and then allowing the eluted proteincomplex to undergo crystallization precipitation with ammonium sulfate30%.

Herein, an eluted solution is a solution fraction after shaking in warmwater for 10 hours or more. An ammonium sulfate precipitate is aprecipitate when the eluted solution is adjusted to a 30% ammoniumsulfate solution. The supernatant is a supernatant fraction obtained bycentrifugal separation of a solution prepared by redissolving theammonium sulfate precipitate in water.

FIG. 4 is SDS-page of a component soluble in an SDS solution containingβ-mercaptoethanol of a wheat gluten dough with or without addition of acommon or table salt found in the description (Role of a Table or CommonSalt in Network Formation of Gluten Protein) of Reiko Urade, in “Foodand Technology”, 2008 December, General remarks.

FIG. 5 shows qualitative analytical results of samples (i) to (vi) usingFourier transform infrared spectrophotometry (FT-IR).

FIG. 6 a shows qualitative analytical results of samples (i) to (iv)using an X-ray microanalyzer EPMA-1600.

FIG. 6 b shows qualitative analytical results of samples (i) to (iv)using an X-ray microanalyzer EPMA-1600.

FIG. 6 c shows qualitative analytical results of samples (i) to (iv)using an X-ray microanalyzer EPMA-1600.

FIG. 6 d shows qualitative analytical results of samples (i) to (iv)using an X-ray microanalyzer EPMA-1600.

FIG. 7 is a graph wherein a yield of a protein complex obtained byleaving a green pea-calcium alginate gel to stand in air for apredetermined time, extracting a protein complex, followed bycross-linking crystallization and further freeze-drying, and anasymmetric oxidation activity (right axis) after 6 hours is monitored inthe second step of the production method of the present invention.

FIG. 8 is a GC chromatogram of a perfume R-1-octen-3-ol (Matsutakeol;98.2% ee) synthesized by reacting a protein complex with a substrateracemic 1-octen-3-ol in the second step.

FIG. 9 a shows a transition of DO (dissolved oxygen) in 100 L jarproduction of a protein complex in the second step of the productionmethod of the present invention.

FIG. 9 b shows a transition of pH in 100 L jar production of a proteincomplex in the second step of the production method of the presentinvention.

FIG. 9 c shows a transition of DO (dissolved oxygen) in 100 L jarproduction of a protein complex in the second step of the productionmethod of the present invention.

FIG. 9 d shows a transition of pH in 100 L jar production of a proteincomplex in the second step of the production method of the presentinvention.

FIG. 9 e shows a transition of DO (dissolved oxygen) in 100 L jarproduction of a protein complex in the second step of the productionmethod of the present invention.

FIG. 9 f shows a transition of pH in 100 L jar production of a proteincomplex in the second step of the production method of the presentinvention,

FIG. 9 g shows a transition of DO (dissolved oxygen) in 100 L jarproduction of a protein complex in the second step of the productionmethod of the present invention.

FIG. 9 h shows a transition of pH in 100 L jar production of a proteincomplex in the second step of the production method of the presentinvention.

FIG. 10 shows a production flow of all production steps of a proteincomplex, and brief description of each step.

FIG. 11 a shows a cell system (Journal of the American Chemical Society,“Crystal structure of the DsbB-DsbA complex reveals a disulfide bondseveration mechanism”, Cell, Vol. 127, Issue 4, 789-801, Nov. 17, 2006:Kenji Inaba et al. (citing the drawing of Medical Institute ofBioregulation, Kyushu University)) for formation of a protein disulfidebond in procaryote (Escherichia coli).

FIG. 11 b shows a cell system (Journal of the American Chemical Society,“Crystal structure of the DsbB-DsbA complex reveals a disulfide bondseveration mechanism”, Cell, Vol. 127, Issue 4, 789-801, Nov. 17, 2006:Kenji Inaba et al. (citing the drawing of Medical Institute ofBioregulation, Kyushu University)) for formation of a protein disulfidebond in eucaryote (yeast endoplasmic reticulum).

FIG. 12 is a block diagram showing kinds and the enzymatic function(reactivity) of a sulfide-based enzyme obtained by cysteine oxidation incells.

FIG. 13 is a graph wherein a yield and an oxidation activity of thoseobtained by freeze-drying cross-linked crystallized protein complexes(CLPCs) obtained in Example 20, followed by ball mill crushing aresummarized.

FIG. 14 is a graph showing the results of tracing of a persistence ratioof R-1-(2-naphthyl)ethanol which is obtained by adding 40 ml of 50 mMTris HCl (pH 8.0) buffer of a racemic 1-(2-naphthyl)ethanolconcentration having a concentration of 10 ppm to a freeze-dried (FD)powder (50 mg, 75 mg, 100 mg, 200 mg, 300 mg) of the protein complexobtained in Example 20 in a 200 ml Erlenmeyer flask, and reacting themthrough warm water shaking in a constant-temperature shaking incubator(40° C., 55 rpm), followed by asymmetric oxidation (every 4 hours) usingGC.

FIG. 15 is a graph showing the results of GC tracing (every 4 hours) ofa persistence ratio of R-1-(2-naphthyl)ethanol which is obtained byadding 40 ml of 50 mM Tris HCl (pH 8.0) buffer containing a racemic1-(2-naphthyl)ethanol having a concentration (10-140 ppm) to afreeze-dried (FD) powder (300 mg) obtained in Example 20 in a 200 mlErlenmeyer flask, and reacting them through warm water shaking in aconstant-temperature shaking incubator (40° C., 55 rpm), followed byasymmetric oxidation.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described more specifically whileoptionally referring to the accompanying drawings. In the followingdescription, “parts” and “percentages” representing a quantitative ratioare by mass unless otherwise specified.

(Protein Complex)

The protein complex of the present invention is characterized in that itcomprises at least a protein and also has at least one peak in a regionof (1,085±50 cm⁻¹) and a region of (1,411±50 cm⁻¹) respectively inFT-IR. Herein, as mentioned below, FT-IR can be measured, for example,by a Micro-ATR method (Ge crystal), using a Fourier-transform infraredspectrometer Magna-750 manufactured by Thermo Fisher Scientific and aninfrared microscope Nic-Plan, under the conditions of a frequencyresolution of 8 cm⁻¹ and integration times of 32 times. (With respect todetails of the FT-IR measurement, for example, the document“Carbohydrate Polymers” 66, 191-197, (2005) (Enzymatically Producednano-ordered short elements containing cellulose I_(β) crystallinedomains) “Noriko Hayashi et al.; Bioconversion Laboratory, Forestry andForest Products Research Institute (FFPRI)”) discloses Examples. Withrespect to the evaluation of absorption wavelength, it is possible torefer to the book “ACADEMIC PRESS; New York and London 1971; ASubsidiory of Harcourt Brace Jovanovich, Publishers” INFRARED SPECTRA OFINORGANIC COMPOUNDS (3800-45 cm⁻¹)” [Richard A. Nyquist and Renald 0.Kagel; Chemistry Physics Research Laboratory, The Dow Chemical CompanyMidland MichiGAn].

(Intensity of IR Peak)

In the protein complex of the present invention, the peak intensity inthe above FT-IR measurement is preferably as follows.

(1) The peak in a region of (1,085±50 cm⁻¹) (hereinafter referred to asa “peak 1”) is preferably a peak having the largest intensity among themeasured IR spectrum, or has a peak intensity of ( 1/10)×I° or more whenthe intensity of a peak having the largest intensity denotes I₀. Thispeak intensity is more preferably (⅕)×I₀ or more (especially (⅓)×I₀ ormore).(2) The peak in a region of (1,411±50 cm⁻¹) (hereinafter referred to asa “peak 2”) is preferably a peak having the largest intensity among themeasured IR spectrum, or has a peak intensity of ( 1/10)×I₀ or more whenthe intensity of a peak having the largest intensity denotes I₀. Thispeak intensity is more preferably (⅕)×I₀ or more (especially (⅓)×I₀ ormore).

(Intensity of Other IR Peaks)

In the protein complex of the present invention, there is no particularlimitation on IR peaks other than the above-mentioned “peak 1” and “peak2”. The protein complex of the present invention may have a peak, forexample, in the below-mentioned region.

In the protein complex of the present invention, evaluation of the aboveFT-IR measurement peak intensity is more preferably as follows.

(1) The peak in a region of (1,085±50 cm⁻¹) (hereinafter referred to asa “peak 1”) is a peak having the largest intensity among the measured IRspectrum and the peak having the second largest intensity exists in aregion of (1,411±50 cm⁻¹) when the intensity of a peak having thelargest intensity denotes I₀ and this peak intensity is more preferably(⅕)×I₀ or more (especially (⅓)×I₀ or more).(2) The peak in a region of (1,411±50 cm⁻¹) (hereinafter referred to asa “peak 2”) is a peak having the second largest intensity among themeasured IR spectrum and the peak having the third largest intensityexists in a region of (1649±50 cm⁻¹) when the intensity of a peak havingthe second largest intensity denotes I₀ and this peak intensity is morepreferably (⅕)×I₀ or more (especially (⅓)×I₀ or more).

(Intensity of Other IR Peak)

Also in the above case, in the protein complex of the present invention,there is no particular limitation on IR peak other than theabove-mentioned “peak 1” and “peak 2”. The protein complex of thepresent invention may have a peak, for example, in the following region.

(Preferred Protein Complex)

The protein complex of the present invention preferably comprises atleast protein and calcium. The protein complex preferably has anactivity in catalyzing an asymmetric oxidation reaction. “Containing atleast protein and calcium” and “Having an activity in catalyzing anasymmetric oxidation reaction” can be suitably confirmed by thebelow-mentioned method.

(Preferred Catalyst of Asymmetric Oxidation Reaction)

A preferred aspect of the asymmetric oxidation reaction of the proteincomplex of the present invention can be suitably represented by anoptical purity (% ee) of R-1-octen-3-ol obtained when a substrateracemic-1-octen-3-ol (Matsutakeol) is reacted under the conditions ofthe below-mentioned Example 12. The protein complex of the presentinvention preferably gives R-1-octen-3-ol with a chemical yield of 85%or more under the conditions of Example 12. Furthermore, this opticalpurity is preferably 95% ee or more (especially 98% ee or less).

(Suitable Characteristics)

The protein complex of the present invention preferably has one or morecharacteristics among the following suitable characteristics.

(1) The protein complex shall further comprise a saccharide.(2) The protein shall be a cross-linked crystallized protein(3) The asymmetric oxidation reaction shall be an oxidation reactionwhich selectively gives one enantiomer of a substrate racemic alcohol.(4) The protein complex shall have an activity which gives aderacemization reaction that allows ketone obtained by the asymmetricoxidation to undergo asymmetric oxidation.

(Estimated Mechanism of the Present Invention)

According to the discovery of the present inventors, it is estimatedthat a suitable protein complex derived from animal and plant isprovided in the present invention for the following reason.

The crude protein derived from animal and plant as the inexpensivematerial is provided with a property wherein (i) an intermoleculardisulfide bond (—S—S—) between cysteine residues (—SH) with each otherin a protein, (ii) intermolecular aggregation (shortening of anintermolecular distance: <6.4 Å) in a protein and (iii) change ofproperty into water solubility occur, and thus not only an active domain(Thioredoxin fold: Cys-X-Y-Cys sequences) is formed, but also a proteincomplex can be suitably eluted by shaking in warm water, byencapsulating in a calcium alginate gel capable of realizing thepresence of a calcium salt and oxygen and then leaving to suitably standin air (air oxidation).

The presence of a dissolved Ca salt and oxygen to be eluted in warmwater has the effect of making a disulfide-bonded protein polymer(protein complex) easily dissolve in an aqueous solution, and they caneasily be extracted by shaking in warm water. A point of an improvementin catalyst activity and an improvement in yield of a protein polymer(protein complex) lies in that the gel beads are suitably left to standin air (air oxidation) and are easily “exudated” in the presence of lowCa ion concentration which dissolves in warm water.

The feature of the production method of the present invention lines inthat a protein complex capable of acting in an enzyme-like manner iseffectively produced at low cost in an environmentally friendly by“reversible idea” such as induction to a suitable disulfide bond, not byimmobilization using a calcium alginate gel, utilizing characteristicsof “protein complex formation” and “exudation” associated with airoxidation of animal and plant proteins.

(Relevant Documents of Estimated Mechanism)

Examples of support of the above-mentioned “estimated mechanism” of thepresent invention include the following documents.

In the document “Food and Technology”, December, pp. 1-9 (2008) (Role ofTable or Common Salt in Network Formation of Gluten Protein), “ReikoUrade; Graduate School of Agriculture, Kyoto University”, it is knownthat a common or table salt and calcium chloride have the effect ofchanging the interaction of a gluten protein, to thereby change theproperty of gliadin and glutenin to the property of soluble in water(water solubility effect) and the effect of enhancing the interactionbetween gluten-constituting proteins, to thereby shorten anintermolecular distance (aggregation effect).

In the effect of shortening the intermolecular distance (aggregationeffect), it is disclosed that the gluten intermolecular distance is 7.7Å in case of adding no salt, while the intermolecular distance isshortened within 6.4 Å in case of adding a salt. Since theintermolecular distance between histidine and iron in an active domainof myoglobin mutant is 5.7 Å, the effect of designing appropriatearrangement of a functional amino acid residue of the active domain canalso be expected as the aggregation effect by addition of a common ortable salt. “Protein, Nucleic acid, Enzyme” 2004, November, Vol. 49, No.14—Molecular Design of Metalloenzyme, Mainly Heme Enzyme—(GraduateSchool of Science, Nagoya University; Yoshihito Watanabe).

There is also reported that it causes a change in the interactionbetween protein molecules when the protein component (glutenin andgliadin) of a wheat flour dough is prepared in the presence of a commonor table salt, and becomes soluble in pure water and exhibitsinsolubility in a saline solution.

Formation of a network structure of a glutenin polymer in wheat glutenis initiated by the addition of water to a wheat flour, and there isknown a function of air oxidation wherein C-terminal and N-terminalcysteine residues of glutenin spontaneously form an intermoleculardisulfide bond (R1-S-S—R2) using oxygen in air. However, latest reports(Non-Patent Document 7) “—Crystal structure of the DsbB-DsbA complexreveals a disulfide bond severation mechanism—Cell 127 789-801 (2006)”(Kenji Inaba et al.; Medical Institute of Bioregulation, KyushuUniversity) disclose that an enzyme for introducing a disulfide bondexists in escherichia coli, and an enzyme (DsbA) capable of oxidizingtwo cysteines of a substrate protein to form a disulfide bond and anenzyme (DsbB) capable of oxidizing the used DsbA to recover an oxidationcapacity functions together with ubiquinone which is a substanceinvolved in generation of activation energy in cells. It is known thatelectrons received by DsbB are donated to ubiquinone (UQ) which is arespiratory chain component and, finally, “oxygen becomes an electronacceptor” through cytochrome oxidase (see FIG. 11 a and FIG. 11 b).

Furthermore, a protein disulfide formation system of procaryote(Escherichia coli) is similar to that of eucaryotic cells (yeastendoplasmic reticulum, etc.) and Erolp exists as a functional homolog ofDsbB in the vicinity of an endoplasmic reticulum membrane in endoplasmicreticulum. Oxidizability for creating a disulfide bond in an Escherichiacoli system is “ubiquinone”, whereas, it is flavin adenine dinucleotide(FAD) in an endoplasmic reticulum system. It is disclosed that aDsbA-DsbB-ubiquinone oxidation system in Escherichia coli andPDI-Erolp-FAD of eukaryote have a mutual relationship of a functionalhomolog (see FIG. 11 a and FIG. 11 b).

In addition, the disulfide bond is a chemical bond which isindispensable for a protein existing on a lot of cell cortex to exactlyform and maintain conformation, and formation and dissociation of thedisulfide bond exert an influence on activity of the prote wherein isimportant for a function of cells, and on existing position in cells,and cope with stress caused by a change in redox environment of cells.

STRUCTURE-FUNCTION STUDIES OF GLUTAREDOXINS AND RELATEDOXIDOREDUCTASRE—] (Tobias H. ElGAn; From THE DEPARTMENT OF BIOSCIENCESAND NUTRITION Karolinska Institutet, Stockholm, Sweden) discloses thatcommon sequence consisting of two cysteines “Thioredoxin fold(Cys-X-Y-Cys)” exist in an enzyme active domain and this site catalyzesformation of the disulfide bond and isomerization, and thus having aredox activity. A hydrophobic amino acid often exists on X and Y ofCys-X-Y-Cys. Cysteine residues become two free thiol groups in areduction type, while they are linked through the disulfide bond in theoxidation type (see the below-mentioned FIG. 11 a and FIG. 11 b).

Components that account for about 70 to 80% of a wheat protein areclassified into gliadin (band existing between 25 kda and 50 kda) suchas albumin, globulin and wheat prolamin, and glutenin consisting of ahigh-molecular weight glutenin (three bands existing between HMW: 75 Kdaand 150 kda) and a low-molecular weight glutenin (band existing betweenLMW: 25 Kda and 50 kda). SDS-page thereof is shown in FIG. 4.

(Targeted Catalyst Activity)

FIG. 12 shows various functions of enzymes formed by intracellularcysteine oxidation as known in the document “Biochemical and BiophysicalResearch Communications” 300, pp. 1-4 (2003) (Multiple roles of cysteinein biocatalysis) “Niroshini M, Giles et al; School of Chemistry,University of Exeyer”. (1) Peptidase, (2) glyceraldehyde 3-phosphatedehydrogenase (GAPDH), (3) alcohol dehydrogenase (ADH), (4) bacterialnitrile hydratase (NHase), (5) ribonucleotide reductase (RNRase), (6)pyruvate formate lyase (PFL), (7) benzylsuccinate synthase (BSS), (8)reduction of glutathione disulfide (GSSG), (9) thioredoxin (Trx), (10)glutathion reductase (GR), (11) thioredoxin reductase (TR), (12) NADHoxidase (Nox), (13) NADH peroXidase (Npx).

As known in Japanese Patent No. 3,294,860 (a process for producing anoptically active alcohol), the first step of extracting a water-solubleprotein from grains or beans; the second step of encapsulating theprotein in a calcium alginate gel; and third step of carrying out anasymmetric oxidation conversion reaction of a substrate using theencapsulated protein as a catalyst are disclosed. As known in JapanesePatent No. 3,683,129 (process for producing an optically activealcohol), the first step of extracting a water-soluble protein selectedfrom egg white and ovalbumin separated from egg white; the second stepof encapsulating the protein in calcium alginate; and third step ofcarrying out an asymmetric oxidation conversion reaction of a substrateusing the encapsulated protein as a catalyst are disclosed.

These inventions differ from the present invention in (1) reference toan active domain formation mechanism in protein molecules to beconstructed by air oxidation, and (2) development to a process forproducing a powdered catalyst which can be stored at a normaltemperature due to removal of moisture after cross-linkingcrystallization, in addition to the above discoveries.

The respective steps constituting the production method of the presentinvention will be described below.

(First Step)

In the extraction of the water-soluble protein in the first step of thepresent invention, grains or beans are crushed, large pieces and husksare removed and the grains and beans crushed powder obtained in thismanner are extracted for 30 minutes or more in water equal to 7-15 timesthe weight of grains or beans at about 20 to 60° C., and preferablyabout 40° C., and a pH of about 6 to 8, and preferably pH of about 7.0.It is most effective to extract for about 45 minutes, and even ifextracted for longer than this, the amount of extract does not change.When it is necessary to adjust pH, pH may be adjusted to theabove-mentioned optimum range using a food-grade acid such as H₂SO₄,HCl, H₃PO₄ or a food-grade alkali such as NaOH. The above-mentionedwater-soluble protein extract or protein curd obtained by separatingfood fiber components from this extract by decanting or centrifugalseparation, is either transferred to the second step as it is, ortransferred to the second step after forming into a powder by spraydrying, freeze-drying or vacuum-drying and then redissolving asnecessary. Seeds (husks (brans, rice brans) and germs (sprouts)) can beobtained by the step of the grains and beans crushed powder. Husks(brans, rice brans), germs (sprouts), leaves (young leaves, sprouts),stems, roots and flowers are dried by freeze-drying (FD), hot-air drying(AD), vacuum-drying and the like, to thereby remove moisture, followedby finely crushing up to 5 μm or less using a ball mill and the like.

The obtained crushed powder can be transferred to the second step as itis. The object of selecting the pH of isoelectric point precipitation isto select the fraction having the largest precipitated amount, and thepH is in the vicinity of 4.5 and 9.5 in case of soy bean, and green peaand buckwheat protein. 5 to 10 times by weight of water are added tothis curd, followed by crushing using a mixer or a stirrer, to therebyprepare a protein slurry, followed by neutralization (pH 6 to 8), tothereby obtain a neutral slurry. After converting this slurry into apowder by spray-drying, freeze-drying (FD) or vacuum-drying in the samemanner as descried above, the powder is redissolved and transferred tothe second step.

However, when it is necessary to treat a large amount of protein, theprotein curd is subjected to an isoelectric point treatment using afood-grade acid such as H₂SO₄, HCl or H₃PO₄, or a food-grade alkali suchas NaOH, followed by separation of the whey by decanting or centrifugalseparation, to thereby obtain a protein curd. This isoelectricprecipitation is performed for the purpose of concentrating of awater-soluble protein, and the same effects as in case of forming into apowder by spray-drying of a water-soluble protein extract, as it is, areexerted even after the treatment.

(Second Step)

In the second step, the process for encapsulating the extracted proteinincludes, for example, an encapsulating method wherein the extractedprotein is either incorporated in the fine matrix of a gel such asalginate, starch, konjak, a polyacrylamide gel or a gel of polyvinylalcohol (matrix type) or coating with a semi-permeable coating(microcapsule type). However, in case of producing a protein complex, anencapsulating method using calcium of alginic acid extracted from kelpis most preferred since it is inexpensive and environmentally friendly,and it is easy to perform an encapsulating operation. The encapsulatedbeads require a warm water temperature of 30° C. to 45° C. so as toelute the protein complex in aqueous solution, and also require oxygensupply so that the dissolved oxygen concentration becomes zero. Also, itis desired to shake at a rotation speed which enables shaking of beads.

(Third Step)

In the third step, in order to allow the protein to undergocrystallization precipitation, ammonium sulfate, polyethylene glycol(PEG), polyethylene glycol/lithium chloride, 2-methyl-2,4-pentadiol,sodium chloride, sodium malonate and the like are exemplified, and anycompound can be used as a crystallization precipitant in the presentinvention. However, it is most advantageous to use inexpensive ammoniumsulfate from the viewpoint of total cost down. It is desired tocrystallize the protein using a protein solution which has aconcentration of 10 to 50 mg/mL, and also there is a method ofconcentration a sample using Centricon, Amicon Ultra and the like.

(Fourth Step)

In the fourth step, in order to cross-link the crystallized precipitatedprotein, glutaraldehyde (hereinafter may be sometimes abbreviated to“GA”), formaldehyde (hereinafter may be sometimes abbreviated to “FA”)and the like are exemplified, and any compounds can be used as across-linking agent in the present invention. Glutaraldehyde is anorganic compound represented by the rational formula OHC(CH₂)₃CHO, andreacts with an 8-amino group as a lysine residue of the protein, andalso reacts with an α-amino group or an SH group, and thus anintermolecular cross-link can be formed. It is not considered that oneglutaraldehyde molecule alone can causes cross-link formation.(Reference document; supervised by Yoshinobu Shigenaka, “Observation ofProtozoan and Test Method (i.e, Genseidoubutu-no-Kansatus-to-JikkenHouhou)” (Kyoritsu Shuppan Co., Ltd., 1998) ISBN 4320053532). GA havetwo aldehyde groups and FA has one aldehyde group, and it becomespossible to be widely utilized as an industrial enzyme reagent in apractical level by cross-linking, for example, (1) it is stable at roomtemperature for a long period (one or more years), (2) it issubstantially 100% active protein (high volume measurement (volumetric)and catalyst productivity), (3) it is easy to recover and recycle, (4)it has high temperature stability and high resistance to an organicsolvent as compared with an enzyme alone, and (5) it has high activityand selectivity as compared with an enzyme alone.

Since GA may have two aldehyde groups, it has a very strong immobilizingforce as compared with FA. While GA has a strong immobilizing force, ithas a drawback such as a very low penetration rate. Since FA has a weakimmobilizing force but shows a penetration rate which is several timeshigher than that of GA, it is possible to make up for mutual weak pointsby mixing a GA immobilization liquid with FA. A GA/FA mixed immobilizingliquid was devised by Karnovsky (1965) (5% GA+4% FA), and name ofKarnovsky is known as “use of dilute Karnovsky” still now. In additionto CA and FA, various additives may be sometimes added in theimmobilizing liquid. Most occasionally, sucrose, glucose, table orcommon salt and the like can be added so as to adjust (increase) anosmotic pressure. With respect to the immobilization time by means ofcross-linking, in case of using a bulk (1 mM or more in thickness),about 2 hours is a minimum time. The temperature of the immobilizingliquid can vary, for example, immobilizing is carried out at roomtemperature or 4° C. (ice water). With respect to reproducibility asimportant, immobilization is preferably carried out at a normaltemperature of from 20° C. to 40° C. When a given temperature isrequired, the temperature can be maintained at about 4° C. by immersings sample bottle containing an immobilizing liquid and a sample in waterwith floating ice. Taking a total operation cost or the like intoconsideration, it is preferred to immobilize at a normal temperature of20° C. to 40° C. After immobilization, residual TA and FA can be washedwith 0.1 M phosphoric acid buffer (about 220 mOsm), or a solutionprepared by adding a 0.1-0.2 M sucrose or glucose to the buffer thereof.

(Fifth Step)

In the fifth step, in order to form into a powder by drying, it ispossible to use (1) hot-air drying (AD), (2) freeze-drying (FD), (3)press drying, (4) compression-drying, (5) air drying and the like in thepresent invention. However, freeze-drying (FD) enables complete removalof moisture, and is advantageous for the subsequent crushing step andalso enhances storability.

(Description of Respective Data)

The respective data shown in the drawings will be described below.

FIG. 1 shows a persistence ratio of a substrate when distilled water isadded to green pea protein-calcium alginate gel beads and a substrate(racemic)-1-(2-naphthyl)ethanol is added at 0 hours, 5 hours, 10 hours,15 hours, 20 hours, 25 hours, 35 hours, 45 hours and 55 hours afterinitiation of blank shaking in the second step.

FIG. 2 a is a graph wherein a ratio (%) of a substrate to a product witha reaction time is monitored when (a) substrates racemic1-(2-naphthyl)ethanol, R-1-(2-naphthyl)ethanol, 2-acetonaphthone areadded in each amount of 50 mg at 10 hour after blank shaking in thesecond step.

FIG. 2 b is a graph wherein a substrate R-1-(2-naphthyl)ethanol (50 mg,75 mg or 100 mg) is added to an aqueous solution (120 ml, 240 ml) at 10hours after blank shaking, beads+distilled water (DW 120 ml) at 10 hoursafter blank shaking, and new beads+aqueous solution (120 ml) at 10 hoursafter blank shaking, respectively, and then an amount (%) of the formedketone is monitored in the second step.

FIG. 2 c is a graph wherein green pea-calcium alginate gel beadsprepared by added dropwise in a different concentration (5 g/L, 7.5 g/L,10 g/L, 15 g/L, 20 g/L, 30 g/L) of calcium chloride is blank-shaken for10 hours and then reacted with a substrate R-1-(2-naphthyl)ethanol, anda protein complex activity is examined in the second step.

FIG. 2 d is a graph wherein green pea-calcium alginate gel beads areprepared by 2 g, 3 g, 4 g and 5 g of a green pea protein andblank-shaken for 10 hours and then and a substrateR-1-(2-naphthyl)ethanol (50 mg) is added and a protein complex activityis examined in the second step.

FIG. 3 is a drawing wherein a protein complex obtained by shaking agreen pea-Ca alginic acid gel in warm water for 10 hours or more isallowed to undergo crystallization precipitation at ammonium sulfate 30%and is further subjected to centrifugal separation (15 minutes, 10,000rpm), to thereby obtain a precipitate, which is then subjected toSDS-page.

SDS-Page

A sample (10 μL) was mixed with 10 μL of 2× sample buffer (0.1 Mtris/HCl pH 6.8, 3% SDS, 10% glycerin, 10% β-mercaptoethanol, 0.1% BPB),followed by heating at 100° C. for 5 minute. The obtained mixture wassubjected to electrophoresis (constant current: 18 mA, electrophoresisbuffer: 25 mM Tris/HCl, 0.19 M Glycine, 0.1% SDS, pH 8.3), together witha molecular weight marker (SDS-PAGE Standard Broad, Bio-Rad), usingSDS-PAGE mini (4-20% Gradient gel, TEFCO Co., Ltd.), and then subjectedto CBB staining (PHastGel Blue R, Amersham Biosciences Corp.).

FIG. 4 is extract of “FIG. 5; Influence of Addition of table or commonsalt on cross-linking efficiency by DST” described in the document “Foodand Technology” December, pp. 1-9 (2008) (Role of Table or Common Saltin Network Formation of Gluten Protein)) “Reiko Urade; Graduate Schoolof Agriculture, Kyoto University” (hereinafter, an explanatory text ismentioned). A dough with or without addition of a common or table saltwas treated with DST, and then a protein was solubilized with an SDSsolution containing β-mercaptoethanol. The solubilized liquid wasultracentrifuged, to thereby separate into a soluble fraction (5) and aninsoluble fraction (P). The insoluble fraction was treated with sodiummetaperiodate. Each sample was separated by SDS polyacrylamide gelelectrophoresis and the protein was stained.

FIG. 5 shows the results analyzed by cross-linked crystallized powders((V) and (vi)), which is obtained by preparing a green pea proteinpowder (i), a sodium alginate powder (ii) and a preparing green peaprotein-calcium alginate gel in accordance with a usual method, omitting(iii) shaking by a constant-temperature shaking incubator, or (iv)protein complex powders ((iii) and (iv)) prepared by stirring in jarfermentor warm water, and the second encapsulating step in the secondstep, and allowing the green pea protein (i) to undergoconstant-temperature shaking (55 rpm, 40° C., 24 hours) in 2% calciumchloride/50 mM Tris HCl buffer ((V) pH 6.0 or (vi) pH 8.0), by FT-IR.

Infrared spectroscopy (FT-IR) was measured at frequency resolution of 8cm⁻¹ and integration times of 39 times by a Micro-ATR method (Gecrystal), using Fourier transform infrared spectrophotometry Magna-750and an infrared microscope Nic-Plan manufactured by Thermo FisherScientific.

In FIG. 5, symbols A to E have the following meanings.

A: vicinity of 1,649 cm⁻¹; a peak peculiar to a peptide bond (—C(═O)—N—)B: vicinity of vicinity of 1,528 cm⁻¹; a peak peculiar to a peptide bond(—C(═O)—N)C: vicinity of 1,411 cm⁻¹; a peak peculiar to a ketone group ofcarboxylate (—C(═O)—O—Na)D: vicinity of 1,122 cm⁻¹ (1,085 cm⁻¹, 1,032 cm⁻¹); a peak peculiar to asugar ether (—C—O—C—)E: vicinity of 1,085 cm⁻¹; a peak peculiar to a ammonium sulfate((NH₄)₂SO₄)

FIG. 6 show the results wherein a protein complex powder (i) prepared byconstant-temperature shaking incubation, cross-linked crystallizedpowders ((ii) and (iii)) obtained from a green pea protein and a 9%calcium chloride/50 mM Tris HCl buffer solution ((ii) pH 6.0 or (iii) pH8.0), and a green pea protein powder (iv) are subjected to X-raymicroanalyzer qualitative analysis, using the same sample as in FIG. 5.X-ray microanalyzer analysis was carried out after coating a surface ofa sample with gold, using EPMA-1600 manufactured by ShimadzuCorporation.

In FIG. 6, the respective samples have the following meanings.

Sample (i): cross-linked crystallized protein complex produced byshaking incubatorSample (ii): 20 mM Ca chloride/50 mM Tris HCl buffer (pH 60)Samples (iii): 20 mM Ca chloride/50 mM Tris HCl buffer (pH 8.0)Sample (iv): green pea protein powder

FIG. 7 is a drawing showing an asymmetric oxidation activity intensity(right axis) after 6 hours of samples obtained by leaving beadsencapsulated in a calcium alginate gel to stand in air for 0 hour, 0.5hour, 1 hour, 3 hours, 5 hours, 7 hours, and then shaking in warm water,to thereby extract a protein complex in the second step, subjecting theprotein complex to cross-linking crystallization, and then freeze-dryingthe protein complex, to thereby form into a powder, and a yield (leftaxis) of the obtained protein complex.

FIG. 8 is GC chromatogram of a perfume R-1-octen-3-ol having an opticalpurity of 98.2% ee, synthesized by encapsulating green pea-calciumalginate gel beads under the same conditions as in FIG. 1 and eluting anaqueous protein complex solution at 10 hours after shaking in the secondstep, and reacting the aqueous protein complex solution with a substrateracemic 1-octen-3-ol (Matsutakeol).

FIG. 9 a to FIG. 9 h (and Table (1)) are drawings and table wherein DO(concentration of dissolved oxygen) and pH monitoring drawing involvedin whether or not oxygen is supplied in a green pea protein powder (1kg)-calcium alginate gel (20 L in total) and addition of water (20 L)using a 100 L jar fermentor, and yield and activity of the obtainedprotein complex are summarized.

FIG. 9 a and FIG. 9 b: Results of first testFIG. 9 c and FIG. 9 d: Results of second testFIG. 9 e and FIG. 9 f: Results of third testFIG. 9 g and FIG. 9 h: Results of fourth test

TABLE 1 Number of extraction Once Twice Three times Four times Reactiontime (hour) 72 48 24 24 D0 control (mg/L) None None 0.25 mg/L 0.25 mg/LTurbidity (NTU) 93.3 292 425 164 Crude yield (g) 16.2 34.2 46.9 26.2Yield amount after FD (g) 5.24 10.05 13.06 6.15 Concentration of Ca(mg/g) 0.53 0.67 0.66 0.54 Asymmetric oxidation C C B B activity(Explanation) Concentration of Ca (FD): The results of the measurementof the concentration of Ca (mg/g) by an acid decomposition method of asample after FD drying using an atomic absorption photometer. C: Only anR-isomer of a racemic 1-(2-naphthyl)ethanol (10 ppm, 40 ml) solution isscarcely asymmetrically oxidized by a “protein complex” (350 mg) within20 hours (the formed ketone is 10% or less). B: Asymmetrically oxidationof only an R-isomer of a racemic 1-(2-naphthyl)ethanol (10 ppm, 40 ml)solution is stopped during asymmetric oxidation by a “protein complex”(350 mg) until 20 hours (the formed ketone is 30% or less). A: Only anR-isomer of a racemic 1-(2-naphthyl)ethanol (10 ppm, 40 ml) solution isasymmetrically oxidized by a “protein complex” (350 mg) within 20 hours(the formed ketone is 40% or more).

FIG. 10 is a process chart of a production flow of a protein complex.

FIG. 11 a shows an intracellular system for formation of a disulfidebond in procaryote (Escherichia coli).

FIG. 11 b shows an intracellular system for formation of a disulfidebond in eucaryote (yeast endoplasmic reticulum).

FIG. 12 is a drawing showing kinds and enzymatic function (reactivity)of a sulfide-based enzyme obtained by cysteine oxidation in cells.

(Protein Complex Having Activity which Catalyzes Asymmetric OxidationReaction)

The protein complex of the present invention is characterized in that itis a product which is formed by air oxidation of a crude protein derivedfrom animal and plant, and also has an enzyme-like activity whichcatalyzes an asymmetric oxidation reaction.

(Process for Measurement of Asymmetric Oxidation Activity)

The activity of the protein complex, which catalyzes an asymmetricoxidation reaction of the present invention can be suitably measured,for example, by the method described hereinafter (6. AsymmetricOxidation Activity in Example 15 “Conditions of Extracting Operation”).

(Process for Purification of Protein Complex)

In the present invention, there is no particular limitation on theprocess for purification of a protein complex. For the purpose ofincreasing a yield of the protein complex, filtration is not required.However, glass beads can be used so as to separate from calcium alginategel beads. In this case, the diameter can be from 40 to 200 μm, andpreferably 200 μm.

The present invention will be described specifically below by way ofExamples, but it should be understood that these are exemplary of theinvention and are not to be considered as limiting.

Example 1 General Process for Producing Cross-Linked CrystallizedProtein Complex Derived from Green Pea

First, in the first step, green peas are crushed, to thereby removehusks, and a green pea protein component dissolved for about 45 minutesin 9 times by weight of distilled water (about 40° C.) at the pH ofabout 7.0 is adjusted to pH 7.0 using an aqueous NaOH solution. The foodfiber of the precipitated component is removed and the protein issubjected to isoelectric point precipitation by bringing thewater-soluble protein portion to alkali conditions (about pH 9.5) oracidic conditions (about pH 4.5). After redissolving the proteinprecipitate moiety with distilled water at pH 7.0, a spray-dryingtreatment is performed on the resulting aqueous green pea proteinsolution (sample concentration: 5.0%) to prepare powdered green peaprotein. In addition, an aqueous 3% sodium alginate solution is preparedby dissolving sodium alginate in aqueous solution under autoclaveconditions and temperature of 121° C. for 20 minutes.

Next, in the second step, 200 ml of distilled water corresponding to 10times the equivalent weight are added to 20 g of a green pea proteinpowder, followed by the addition of 200 ml of an aqueous 3% sodiumalginate solution corresponding to 1.0 times the equivalent weight andstirring until uniform. The resulting green pea-sodium alginate mixedsolution is added dropwise in an aqueous 4% calcium chloride solution,to thereby prepare green pea protein-containing calcium alginate gelbeads in an immobilized state. The beads were left to stand in air for 5hours. The green pea protein-containing calcium alginate gel beads waswashed with distilled water, to thereby remove an aqueous calciumchloride solution, and distilled water (400 ml) corresponding to 20times the equivalent weight of the used green pea protein powder wasadded as an extract solution. Constant-temperature shaking incubation(40° C., 55 rpm) was carried out for 2 days and the water-solublefraction was separated and recovered, and then beads were further shakenfor one day, to thereby recover the objective solution in the samemanner. After shaking for additional one day, the objective solution canbe separated and recovered. The protein complex comprised in therecovered reaction solution was subsequently allowed to undergocrystallization precipitation in the third step.

In the third step, the separated and recovered solution was adjusted tosaturated 30% ammonium sulfate for the purpose of precipitation of aslightly water-soluble component, and then left to stand for 20 hours ormore. The protein complex was not only eluted easily from beads in thepresence of calcium chloride, but also it became water insoluble in thepresence of a saturated 30% ammonium sulfate solution and thenaggregated and precipitated.

In the fourth step, a commercially available 25% glutaraldehyde solutionwas further added to the precipitated and aggregated crystallizedprotein complex so that the concentration of the solution becomes 0.25%,and the solution was left to stand for 2 hours or more (preferably 10hours or more). The obtained protein complex could be easily separatedfrom the solution by decantation using a cold centrifuge (10,000 rpm, 15min, 10° C.).

In the fifth step (freeze-drying (FD)), the protein complex of theprecipitate was cooled to −50° C. and left to stand for 1 hour. Afteradjusting to a vacuum state of about 0.1 mbar or less, the temperaturewas raised from 50° C. to 50° C., to thereby cause sublimation andremoval of moisture. A series of required FD time was about 20 hours.

In the sixth step, the protein complex wherein moisture could be removedby freeze-drying (FD) could be converted into a powdered catalystcapable of being stored at normal temperature by the subsequent ballmill crushing.

Example 2 Examination of Substrate Specificity of Protein ComplexDerived from Green Pea Protein (PP), Soy Bean Protein (SP) and WheatProtein (WP) in Second Step

In the second step, with respect to a crude protein derived from greenpea protein (PP), soy bean protein (SP) and wheat protein (WP), seedsare crushed and water is added. After 1 hour, a bean curd lee fractionof the precipitate is removed and an aqueous solution fraction isspray-dried, to thereby obtain 20 g of a powder. In accordance with ausual method, 200 ml of distilled water corresponding to 10 times theequivalent weight is added and 200 ml of an aqueous 3% sodium alginatesolution is added corresponding to 1 time the equivalent weight,followed by stirring until uniform. The obtained green pea-sodiumalginate mixed solution is added dropwise in an aqueous 4.0% calciumchloride solution, to thereby obtain green pea protein-containingcalcium alginate gel beads. To the thus obtained beads, distilled water(400 ml) of a green pea protein powder corresponding to 20 times theequivalent weight is added as the reaction solution. After shaking for10 hours or more using a constant-temperature shaking incubator underthe conditions of a temperature setting of 40° C. and the number ofshaking of 55 rpm, a substrate conversion reaction was carried out. Asthe substrate, 1-(2-naphthyl)ethanol (naphthyl/1) and phenylethanolwherein chlorine (Cl-/2a, 2b), bromine (Br-/3a, 3b), fluorine (F-/4a,4b), methyl (Me-/5a, 5b), methoxy (MeO-/6a, 6b) and nitro (NO2-/7a, 7b)are substituted on the meta- and para-positions were used. As a result,the reaction time, product, optical purity and chemical yield aresummarized in the table below.

(Substrate Specificity of Meta-Substituted Substrate of Protein ComplexDerived from Green Pea Protein (PP), Soy Bean Protein (SP), WheatProtein (WP) in Second Step)

TABLE 2 (Substrate Specificity of Meta-Substituted Substrate of Grains -Beans Proteins in Second Step) Substrate (±)-ArCH(OH)R Reaction PlantProducts Ar R time origin Comp OP/% e.e. CY/% 1 2-Naphthyl Me 15 PP S-1≧99 50 2a 3-ClC6H4 Me 24 PP S-2a ≧99 50 3a 3-BrC6H4 Me 36 PP S-3a ≧99 494a 3-FCC6H4 Me — PP Not resolved! 5a 3-MeC6H4 Me 29 PP S-5a ≧99 50 6a3-Me0C6H4 Me 36 PP S-6a ≧99 50 1 2-Naphthyl Me 25 SP S-1 ≧99 49 2a3-ClC6H4 Me 25 SP S-2a ≧99 50 3a 3-BrC6H4 Me 25 SP S-3a ≧99 49 4a3-FCC6H4 Me — SP Not resolved! 5a 3-MeC6H4 Me 45 SP S-5a ≧99 50 6a3-Me0C6H4 Me 30 SP S-6a ≧99 50 1 2-Naphthyl Me — WP Not resolved! 2a3-ClC6H4 Me 25 WP S-2a ≧99 49 3a 3-BrC6H4 Me 18 WP S-3a ≧99 50 4a3-FCC6H4 Me 76 WP S-4a ≧99 50 5a 3-MeC6H4 Me 38 WP S-5a ≧99 49 6a3-Me0C6H4 Me 53 WP S-6a ≧99 50 Supplement: Comp: compound, OP/% e.e.:optical purity, CY/%: chemical yield

As is apparent from the above results, a protein complex derived fromgreen pea protein (PP), soy bean protein (SP) and wheat protein (WP) wassuitably eluted from a gel and, at the same time, an R-isomer of racemicphenyl ethanols of meta-substituted chlorine (Cl-/2a), bromine (Br-/3a),fluorine (F-/4a), methyl (Me-/5a), methoxy (MeO-/6a) and nitro (NO2-/7a)as the substrate was selectively oxidized, and an S-isomer alcohol wasobtained with a chemical yield of about 50 and an optical purity of 99%e.e. or more.

(Examination of Substrate Specificity of Para-Substituted Substrate ofProtein Complex Derived from Green Pea Protein (PP), Soy Bean Protein(SP) and Wheat Protein (WP) in Second Step)

TABLE 3 (Substrate Specificity of Para-Substituted Substrate of Grains -Beans Proteins in Second Step) Substrate (±)-ArCH(OH)R Reaction PlantProducts Ar R time origin Comp OP/% e.e. CY/% 2b 4-ClC6H4 Me 15 PP S-2b≧99 50 3b 4-BrC6H4 Me 24 PP S-3b ≧99 50 4b 4-FC6H4 Me 20 PP S-4b ≧99 505b 4-MeC6H4 Me 19 PP S-5b ≧99 49 6b 4-Me06H4 Me 19 PP S-6b ≧99 50 7b4-NO2C6H4 Me 45 PP S-7b ≧99 50 2b 4-ClC6H4 Me 19 SP S-2b ≧99 50 3b4-BrC6H4 Me 19 SP S-3b ≧99 49 4b 4-FC6H4 Me 34 SP S-4b ≧99 50 5b4-MeC6H4 Me 25 SP S-5b ≧99 50 6b 4-Me06H4 Me 18 SP S-6b ≧99 50 7b4-NO2C6H4 Me 30 SP S-7b ≧99 50 2b 4-ClC6H4 Me 23 WP S-2b ≧99 50 3b4-BrC6H4 Me 29 WP S-3b ≧99 50 4b 4-FC6H4 Me 18 WP S-4b ≧99 50 5b4-MeC6H4 Me 29 WP S-5b ≧99 50 6b 4-Me06H4 Me 15 WP S-6b ≧99 50 7b4-NO2C6H4 Me WP Not resolved! Supplement: Comp: compound, OP/% e.e.:optical purity, CY/%: chemical yield

As is apparent from the above results, a protein complex derived fromgreen pea protein (PP), soy bean protein (SP) and wheat protein (WP) wassuitably eluted from a gel and, at the same time, an R-isomer of racemicphenyl ethanols of a para-substituted chlorine (Cl-/2b), bromine(Br-/3b), fluorine (F-/4b), methyl (Me-/5b), methoxy (MeO-/6b) and nitro(NO2-/7b) as the substrate was selectively oxidized, and an S-isomeralcohol was obtained with a chemical yield of 50 and an optical purityof 99% e.e. or more.

Example 3 Examination of Substrate Specificity of Protein ComplexDerived from Tissue Material Derived from Other Plants in Second Step

In the second step, for the purpose of confirming elution of an activityof a protein complex, which catalyzes an asymmetric oxidation reactionfrom seeds, leaves, stems, roots, flowers and fruits of grasses andweeds such as young wheat leaves (YWL), young barley leaves (YBL),Artemisia vulgaris indica (AVI) leaves, wheat brans (WB), wakame(Undaria) (WS), carrot (C) and pumpkin (P) further included therein,these grasses and weeds were washed by immersing in warm water at 60° C.or more for 10 minutes, optionally sliced thinly, freeze-dried (FD) andthen finely crushed using a ball mill. In accordance with a usualmethod, 200 ml of distilled water corresponding to 10 times theequivalent weight was added to 20 g of these plant material powders, and200 ml of an aqueous 3% sodium alginate solution corresponding to 1 timethe equivalent weight was added, followed stirring until uniform. Theobtained plant tissue-containing sodium alginate mixed solution wasadded dropwise in an aqueous 4.0% calcium chloride solution, to therebyobtain plant tissue-containing calcium alginate gel beads. To the beads,distilled water (400 ml) corresponding to 20 times the equivalent weightof the plant tissue-containing powder was added as the reactionsolution. After shaking for 10 hours or more under the conditions ofconstant-temperature shaking temperature setting of 40° C. and thenumber of shaking of 55 rpm, a substrate conversion reaction was carriedout. As the substrate, phenylethanols wherein bromine (Br-/1m, 1p),chlorine (Cl-/2m, 2p), fluorine (F-/3m, 3p), methyl (Me-/4m, 4p),methoxy (MeO-/5m, 5p) and 1-(2-naphthyl)ethanol (naphthyl/6) have beensubstituted on the meta- and para-positions were reacted. With respectto the obtained results, the reaction time, product, optical purity andchemical yield are summarized in the table below.

(Examination of Substrate Specificity of Meta-Substituted Substrate ofProtein Complex Derived from Young Wheat Leaves, Young Barley Leaves andWheat Brans in Second Step)

TABLE 4 (Substrate Specificity of Meta-Substituted Substrate of ProteinComplex derived from Young Wheat Leaves, Young Barley Leaves and WheatBrans in Second Step) Substrate Reaction Plant Products Ar time originComp OP/% e.e. CY/% 1m 3-BrC6H4 14 YWL S-1m ≧99 50 3-BrC6H4 12 YBL S-1m≧99 50 2m 3-ClC6H4 26 YWL S-2m ≧99 50 3-ClC6H4 31 YBL S-2m ≧99 50 3m3-FC6H4 85 WB S-3m ≧99 50 3-FC6H4 34 YBL S-3m ≧99 50 4m 3-MeC6H4 39 WBS-4m ≧99 50 3-MeC6H4 26 YWL S-4m ≧99 50 3-MeC6H4 49 YBL S-4m ≧99 50 5m3-Me0C6H4 63 WB S-5m ≧99 50 3-Me0C6H4 26 YWL S-5m ≧99 50 3-Me0C6H4 29YBL S-5m ≧99 25 6 2-naphthyl 12 YWL S-6 ≧99 50 2-naphthyl 10 YBL S-6 ≧9950 Supplement: Comp: compound, OP/% e.e.: optical purity, CY/%: chemicalyield

As is apparent from the above results, a protein complex derived fromyoung wheat leaves (YWL), young barley leaves (YBL) and wheat brans (WB)was suitably eluted from the gel and, at the same time, an R-isomer ofracemic phenyl ethanols of meta-substituted bromine (Br-/1m), chlorine(Cl-/2m), fluorine (F-/3m), methyl (Me-/4m), methoxy (MeO-/5m) and1-(2-naphthyl)ethanol (naphthyl/6) as the substrate was selectivelyoxidized, and an S-isomer alcohol was obtained with a chemical yield of50 and an optical purity of 99% e.e. or more.

(Examination of Substrate Specificity Para-Substituted Substrate ofProtein Complex Derived from Young Wheat Leaves, Young Barley Leaves andWheat Brans in Second Step)

TABLE 5 (Substrate Specificity of Para-Substituted Substrate of ProteinComplex derived from Young Wheat Leaves, Young Barley Leaves and WheatBrans in Second Step) Substrate Reaction Plant Products Ar time originComp OP/% e.e. CY/% 1p 4-BrC6H4 35 WB S-1p ≧99 50 4-BrC6H4 16 YWL S-1p≧99 50 4-BrC6H4 23 YBL S-1p ≧99 50 2p 4-ClC6H4 23 YWL S-2p ≧99 504-ClC6H4 21 YBL S-2p ≧99 50 3p 4-FC6H4 36 YWL S-3p ≧99 50 4-FC6H4 29 YBLS-3p ≧99 50 4p 4-MeC6H4 62 WB S-4p ≧99 50 4-MeC6H4 20 YWL S-4p ≧99 504-MeC6H4 21 YBL S-4p ≧99 50 5p 4-Me0C6H4 21 WB S-5p ≧99 28 4-Me0C6H4 16YWL S-5p ≧99 50 4-Me0C6H4 17 YBL S-5p ≧99 50 Supplement: Comp: compound,OP/% e.e.: optical purity, CY/%: chemical yield

As is apparent from the above results, a protein complex derived fromyoung wheat leaves (YWL), young barley leaves (YBL) and wheat brans (WB)was suitably eluted from the gel and, at the same time, an R-isomer ofracemic phenyl ethanols of para-substituted bromine (Br-/1p), chlorine(Cl-/2p), fluorine (F-/3p), methyl (Me-/4p), methoxy (MeO-/5p) and1-(2-naphthyl)ethanol (naphthyl/6) as the substrate was selectivelyoxidized, and an S-isomer alcohol was obtained with a chemical yield ofabout 50 and an optical purity of 99% e.e. or more.

(Examination of Substrate Specificity of Protein Complex Derived fromArtemisia vulgaris Indica Leaves, Wakame (Undaria), Carrot and Pumpkinin Second Step)

TABLE 6 (Substrate Specificity of Para-Substituted Substrate of ProteinComplex derived from Artemisia Vulgaris Indica Leaves, Wakame (Undaria),Carrot and Pumpkin) Substrate Reaction Plant Products Ar time originComp OP/% e.e. CY/% 1p 4-BrC6H4 30 P S-1P ≧99 50 2p 4-ClC6H4 38 C S-2P≧99 41 4-ClC6H4 30 P S-2P ≧99 50 3p 4-FC6H4 3P Not resolved! 4p 4-MeC6H447 WS S-4P ≧99 21 4-MeC6H4 50 P S-4P ≧99 50 5p 4-Me0C6H4 47 WS S-5P ≧9932 4-Me0C6H4 21 P S-5P ≧99 25 6 2-naphthyl 31 AVI R-6 ≧99 50 Supplement:Comp: compound, OP/% e.e.: optical purity, CY/%: chemical yield

As is apparent from the above results, a protein complex derived fromArtemisia vulgaris indica (AVI) leaves, wakame (Undaria) (WS), carrot(C) and pumpkin (P) was suitably eluted from the gel and, at the sametime, an R-isomer of racemic phenyl ethanols of para-substituted bromine(Br-/1p), chlorine (Cl-/2p), fluorine (F-/3p), methyl (Me-/4p), methoxy(MeO-/5p) and 1-(2-naphthyl)ethanol (naphthyl/6) as the substrate wasselectively oxidized, and an S-isomer alcohol was obtained with achemical yield of 20 to 50 and an optical purity of 99% e.e. or more.

Example 4 Examination of Substrate Specificity of Para-SubstitutedSubstrate of Protein Complex Derived from Egg White Albumin in SecondStep

In the second step, for the purpose of measuring an activity of aprotein complex produced from ovalbumin separated from egg white, eggwhite was separated from chicken egg and ovalbumin was separated byammonium sulfate precipitation. After dissolving in water so that theconcentration of ovalbumin becomes 0.1 to 0.5%, a spray-drying treatmentwas carried out, to thereby prepare an egg white albumin powder. Inaccordance with a usual method, 200 ml of distilled water correspondingto 10 times the equivalent weight was added to 20 g of an egg whitealbumin powder and 200 ml of an aqueous 3% sodium alginate solutioncorresponding to 1 time the equivalent weight was added, followed bystirring until uniform. The obtained egg white albumin-sodium alginatemixed solution was added dropwise in an aqueous 4.0% calcium chloridesolution, to thereby obtain egg white albumin-calcium alginate gelbeads. To the obtained beads, distilled water (400 ml) corresponding to20 times the equivalent weight of the egg white albumin powder was addedas the reaction solution. After shaking for 10 hours or more at aconstant-temperature shaking incubation setting of 40° C. and the numberof shaking of 55 rpm, a substrate conversion reaction was carried out.As the substrate, racemic phenyl ethanols wherein bromine (Br-/1),chlorine “Cl-/2), methoxy (Me0-/5) and 1-(2-naphthyl)ethanol(naphthyl/6) were substituted on the para-position was reacted. Theresults of the obtained reaction time, product, optical purity andchemical yield are summarized in Table 7 below.

(Examination of Position Specificity of Para-Substituted Substrate ofProtein Complex Derived from Egg White Albumin in Second Step)

TABLE 7 (Substrate Specificity of Para-Substituted Substrate by EggWhite Albumin in Second Step) Substrate Reaction Plant Products Ar timeorigin Comp OP/% e.e. CY/% 1p 4-BrC6H4 24 I0A R-1P 86.6 27 2p 4-ClC6H424 I0A R-2P 96.4 26 5p 4-Me0C6H4 24 I0A R-5P 99.8 26 6 2-naphthyl 24 I0AR-6 85.8 24 Supplement: Comp: compound, OP/% e.e.: optical purity, CY/%:chemical yield

As is apparent from the above results, a protein complex derived fromegg white albumin was suitably eluted from the gel and, at the sametime, an S-isomer of racemic phenyl ethanols of substratepara-substituted bromine (Br-/1), chlorine (Cl-/2), methoxy (MeO-/5) and1-(2-naphthyl)ethanol (naphthyl/6) was selectively oxidized, to therebyobtain an R-isomer alcohol with a chemical yield of 26% and an opticalpurity of 85 to 95% e.e. As is apparent from the results, an S-isomeralcohol (99% e.e.) is obtained in case of a protein complex derived froma crude protein derived from plant resource, while a reverse R-isomeralcohol (85 to 95% e.e.) is obtained in case of a protein complexderived from egg white albumin. Thus, it becomes possible to properlyuse both enantiomers by properly using animals and plants.

Example 5 Reason why Blank Shaking is Carried Out inConstant-Temperature Shaker for 10 Hours in Second Step

FIG. 1 shows a graph showing a persistence ratio (%) of a substrate withan asymmetric oxidation activity of a protein complex derived from agreen pea protein thereof when 120 ml of distilled water is added to agreen pea protein-calcium alginate gel beads prepared by dissolving 4 gof a green pea protein in 40 ml of distilled water, further adding 3%sodium alginate (40 ml), followed by stirring until uniform, and addingdropwise the obtained solution in an aqueous 4.0% calcium chloridesolution, and then a substrate R-1-(2-naphthyl)ethanol is added at 0hour, 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 35 hours, 45hours and 55 hours after initiation of blank shaking at aconstant-temperature shaker temperature setting of 40° C. and the numberof shaking of 55 rpm in the second step.

As is apparent from the above results, with respect to the asymmetricoxidation reaction of the protein complex derived from the green peaprotein, the protein complex is eluted from the gel at 10 hours afterblank shaking and, at the same time, it is suitably reacted with thesubstrate R-1-(2-naphthyl)ethanol.

Example 6a Second Step, Examination of Substrate Specificity

FIG. 2 a is a graph wherein a ratio (%) of a substrate to a product witha reaction time is monitored for the purpose of examining substratespecificity when substrates such as racemic 1-(2-naphthyl)ethanol,R-1-(2-naphthyl)ethanol and 2-acetonaphthone are added in each amount of50 mg at 10 hours after initiation of shaking under the same conditionsas in FIG. 1. As is apparent from the above results, the activity whichappear at 10 hours after blank shaking does not causes oxidation of theS-isomer alcohol and allows only an R-isomer of the substrate racemic1-(2-naphthyl)ethanol to undergo stereoselective asymmetric oxidation.

Example 6b Second Step, Examination of Reaction Position

FIG. 2 b is a graph wherein a substrate R-1-(2-naphthyl)ethanol (50 mg,75 mg or 100 mg) is added to an aqueous solution (120 ml) at 10 hoursafter blank shaking, an aqueous solution (240 ml) at 10 hours afterblank shaking, an aqueous solution at 10 hours after blankshaking+distilled water (DW: 120 ml), and new beads+an aqueous solutionat 10 hours (120 ml) after blank shaking, respectively, after preparinga green pea-sodium alginate under the same conditions as in FIG. 1, andthen an asymmetric oxidation catalyst is monitored in the second step.

As is apparent from the above results, the protein complex, whichappears at 10 hours after blank shaking, has the area where the reactionoccurs, which does not exist in beads but in an aqueous solution, andsuitably reacts only with an R-isomer of 1-(2-naphthyl)ethanol.

Example 6c Second Step, Effect of Concentration of Encapsulated GelledCalcium Chloride Exerted on Activity

FIG. 2 c is a graph wherein a green pea-sodium alginate mixed liquid isprepared under the same conditions as in FIG. 1 and added dropwise in asolution each having a different concentration of calcium chloride (5g/L, 7.5 g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L) and, after gelling andblank shaking for 10 hours under the same conditions, a substrateR-1-(2-naphthyl)ethanol is added in the second step, and then adifference in activity intensity of the protein complex, which exerts aninfluence on the concentration of calcium chloride, is summarized.

As is apparent from the above results, as the concentration of calciumchloride becomes lower, formation and elution of a protein complex areefficient and asymmetric oxidation activity increases. In contrast, asthe concentration of calcium chloride becomes higher, the proteincomplex may be hardly eluted. On the other hand, a difference inactivity was scarcely recognized when the concentration of calciumchloride is 10 g/L, 15 g/L or 20 g/L.

Example 6d Second Step, Optimization of Amount of Green Pea Protein

FIG. 2 d is a graph showing an influence of the amount (2 g, 3 g, 4 g, 5g) of a green pea protein at the time of preparation of a green peaalginic acid gel in the second step on activity. In this case, a greenpea-calcium alginate gel was prepared in the concentration of calciumchloride of 10 g/L, and an influence of a protein complex obtained at 10hours after blank shaking exerted on a substrate R-1-(2-naphthyl)ethanol(50 mg) was shown in the drawing.

With respect to the conditions of formation and extraction of a suitableprotein complex, the amount of a green pea protein is 4 g. As a result,when the amount of the green pea protein is from 2 g to 5 g, adifference in protein complex involved in activity was scarcelyrecognized.

Example 7 Second Step, Examination of Synthesis of R-1-Octen-3-Ol asPerfume)

FIG. 8 is a diagram wherein encapsulating in a green pea-calciumalginate gel was carried out under the same conditions as in FIG. 1 inthe second step and the effect involved in resolution of a proteincomplex at 10 hours after blank shaking of a substrate racemic1-octen-3-ol is examined.

As is apparent from the above results, with respect to the catalystaction of the protein complex, a chromatographic peak of the formedketone is 2% or less and the chemical yield is 85% or more, and thus GCchromatogram of R-1-octen-3-ol (98.2% e.e.) shown in FIG. 8 is obtainedby deracemization (continuous recycling is examined in thebelow-mentioned Example 12 and Table 11).

Example 8 Examination of Extract of Protein Complex and SuitableConcentration of Ammonium Sulfate in Third Step)

A protein complex solution (40 ml) obtained under the conditions ofExample 1 was transferred in a 50 mL plastic centrifuge tube, and (1)8.8 g of each ammonium sulfate was added, to thereby adjust to saturated30% and, after being left to stand for 20 hours or more, (2) centrifugalseparation (10,000 rpm, 15 min) was carried out and, after removing theammonium sulfate solution through decantation, or (3) 10 mlracemic-1-(2-naphthyl)ethanol (3 ppm) dissolved in 50 mM-Tris HCl buffer(pH 8.0) was added, asymmetric oxidation activity was measured. (4) Thesolution fraction after decantation was further adjusted to saturated40% by adding 3.1 g of ammonium sulfate and, after being left to standfor 20 hours or more, asymmetric oxidation activity of the precipitateobtained by centrifugal separation was determined in the same manner.The solution fraction after decantation was further adjusted tosaturated 50% by adding 3.15 g of ammonium sulfate and, after stillstanding and further centrifugal separation, oxidation activity of theprecipitate was determined in the same manner. The solution fractionafter decantation was adjusted to saturated 60% by adding 3.3 g ofammonium sulfate and, after centrifugal separation and further stillstanding, oxidation activity was determined in the same manner. Thesolution fraction after decantation was adjusted to saturated 70% byadding 3.45 g of ammonium sulfate and, after centrifugal separation andfurther still standing, oxidation activity was determined in the samemanner. The results are shown below.

TABLE 8 (Results at 30 Hours after Reaction) (Results at 30 Hours afterReaction) Ketone oxide R-Alcohol S-Alcohol Saturated 30% 3.23 43.3553.42 (8.8 g) Saturated 40% 0.36 47.26 52.38 (3.1 g) Saturated 50% 0.9645.27 53.77 (3.15 g) Saturated 60% 0.48 46.90 52.61 (3.3 g) Saturated70% — 47.50 52.50 (3.45 g) Numerical value = GC % ratio Numerical value= GC % ratio

As is apparent from the above results, an optimum concentration ofammonium sulfate for crystallization of the protein complex of the thirdstep is saturated 30%. It was possible to estimate that the obtainedprotein complex is a water-insoluble gluten-like protein and has aproperty changed to water solubility in the presence of calcium, andthus making it possible to precipitate under weak water solubilitycondition of saturated 30%.

Example 9 Concentration of Substrate to be Treated by CrystallizedProtein Complex in Third Step)

The aqueous crystallized protein complex solution obtained under theconditions of Example 1 was transferred in a 50 mL plastic centrifugetube, and (1) 8.8 g of each ammonium sulfate was added, to therebyadjust to saturated 30% and, after being left to stand, (2) centrifugalseparation (10,000 rpm, 15 min) was carried out and, after decantation,to the crystallized protein complex obtained as a precipitate, (3) 10 mLof a racemic-1-(2-naphthyl)ethanol solution (having a concentration of 1ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm) dissolved in 50 mM-Tris HCl buffer (pH8.0) was added, followed by examination.

TABLE 9 (Results at 30 Hours after Reaction) (Results at 30 Hours afterReaction) After 30 hours Ketone oxide R-Alcohol S-Alcohol Saturated 30%,15.16 14.91 69.93 Substrate 1 ppm Saturated 30%, 8.28 19.30 72.42Substrate 2 ppm Saturated 30%, 14.90 15.58 69.51 Substrate 3 ppmSaturated 30%, 9.77 22.06 68.17 Substrate 4 ppm Saturated 30%, 12.5321.05 66.42 Substrate 5 ppm Numerical value = GC % ratio Numerical value= GC % ratio

As is apparent from the above results, the concentration of thesubstrate racemic 1-(2-naphthyl)ethanol, which enables oxidation ofabout 50 mg/50 ml centrifuge tube of the crystallized protein complexcontaining moisture obtained by the third step is from 1 ppm to 5 ppm/10ml and there is no remarkable difference, and asymmetric oxidation canbe carried out.

Example 10 Examination of Extract of Crystallized Protein Complex,Glutaraldehyde Cross-Linking Concentration of 0.1% and 1.0% in FourthStep

The protein complex solution (40 ml) obtained under the conditions ofExample 1 was transferred in a 50 mL plastic centrifuge tube, and (1)8.8 g of ammonium sulfate was added, to thereby adjust to saturated 30%and, after being left to stand for 20 hours or more, (2) a 25%glutaraldehyde solution was added so that the concentration becomes 0.1%(0.32 ml/40 ml) and 1.0% (1.6 ml/40 ml), respectively and, after beingleft to stand for 10 hours or more, (3) centrifugal separation (10.000rpm, 15 min) was carried out, to thereby remove an ammoniumsulfate-glutaraldehyde solution through decantation as the supernatant.With respect to the protein complex of the precipitate, 10 ml of racemic1-(2-naphthyl)ethanol (in terms of the concentration of 3 ppm) dissolvedin 50 mM Tris-HCl buffer (pH 8.0) was added and activity of asymmetricoxidation was confirmed.

TABLE 10 (Results at 30 Hours and 60 Hours after Reaction) Ketone oxideR-Alcohol S-Alcohol After 30 hours Saturated 30%, 21.75 8.67 69.55Substrate 3 ppm, GA concentration 0.1% Saturated 30%, 4.76 27.02 68.23Substrate 3 ppm, GA concentration 1.0% After 60 hours Saturated 30%,8.95 0.0 91.05 Substrate 3 ppm, GA concentration 0.1% Saturated 30%,4.20 22.36 78.58 Substrate 3 ppm, GA concentration 1.0% Numerical value= GC % ratio Numerical value = GC % ratio

As is apparent from the above results, the concentration of cross-likedGA of the crystallized protein complex of the fourth step is 0.1% or1.0% and exhibits oxidation activity in both cases. However, R-alcoholasymmetric oxidation activity progressed more suitably when theconcentration is 0.1% as compared with the case of 1.0%. It could beconfirmed that the deracemization reaction is likely to occur as theconcentration becomes closer to 0.1%.

Example 11 Examination of Continuous Recycling Involved in ProteinComplex Powder in Fourth Step)

The protein complex solution (40 ml) obtained under the conditions ofExample 1 was transferred to each of four 50 mL plastic centrifugetubes, respectively, and (1) 8.8 g of each ammonium sulfate was added,to thereby adjust to saturated 30% and, after being left to stand for awhole day and night, (2) 0.32 ml/40 ml of a 25% glutaraldehyde solutionwas added so that the concentration becomes 0.1%, respectively, followedby being left to stand for 2 to 4 hours. The ammoniumsulfate-glutaraldehyde solution as the supernatant was (3) subjected tocentrifugal separation (10.000 rpm, 15 min) and removed throughdecantation, and the objective precipitate protein complex was furthercrushed after freeze-drying (FD). The protein complex powder (4) (358mg) was dissolved in 3 ppm racemic 1-(2-naphthyl)ethanol-50 mM Tris-HClbuffer (pH 8.0. 40 mL) and then activity was measured. Continuousrecycling was carried out after 9 hours and 15 hours. After centrifugalseparation, the precipitate was recovered and 3 ppm racemic1-(2-naphthyl)ethanol-50 mM Tris-HCl buffer (pH 8.0. 40 mL) was newlyadded and examination was carried out by this repeating operation. Theresults are as follows.

TABLE 11 (Results of Continuous Recycling of Protein Complex) (Resultsof Continuous Recycling of Protein Complex) Continuous recycling Ketoneoxide R-Alcohol S-Alcohol First time: 25 hours after 42.65 0 57.35Second time: 9 hours after 32.13 7.39 60.48 Third time: 15 hours after38.98 4.97 56.05 Fourth time: 9 hours after 35.66 10.28 54.06 Fifthtime: 15 hours after 35.04 9.85 55.11 Sixth time: 9 hours after 37.926.58 55.50 Seventh time: 15 hours after 46.64 0 53.36 Eighth time: 9hours after 45.98 0 54.02 Ninth time: 15 hours after 43.10 3.90 53.00Tenth time: 9 hours after 47.19 1.84 50.97 Eleventh time: 15 hours after48.75 0 51.25 Twelfth time: 9 hours after 41.06 5.48 53.45 Thirteenthtime: 15 hours after 44.47 0 55.53 Fourteenth time: 9 hours after 35.8110.88 53.31 Fifteenth time: 15 hours after 36.57 6.18 57.26 Nineteenthtime: 9 hours after 36.51 9.59 53.90 Numerical value = GC % ratioNumerical value = GC % ratio

As is apparent from the results of confirmation of possibility ofcontinuous recycling involved in the protein complex powder of thefourth step, asymmetric oxidation activity can be maintained at least 16times (8 days) and recycling can be carried out.

Example 12 Synthesis of Perfume (R-1-Octen-3-01) Involved in ContinuousRecycling of Protein Complex Powder in Fourth Step

A trial of an asymmetric oxidation reaction was made in a 500 mlErlenmeyer flask by adding 150 ml of 50 mM Tris-HCl (pH 8.0) buffercontaining racemic 1-octen-3-ol having a concentration of 10 ppm to 5 gof a freeze-dried (FD) powder of the protein complex obtained in Example11. After 30 hours or 60 hours, only the supernatant was recovered bycentrifugal separation (10,000 rpm, 15 min) of 5 ml of the reactionsolution. After extracting the substrate with diethylether, GCmeasurement was carried out. The operation of continuous recyclingleaded to an optical purity and a yield in the same manner as in Example11. The reaction was terminated after 60 hours and the supernatantmoiety obtained after centrifugal separation was extracted with diethylether, washed with saturated saline solution and then isolated andpurified by silica gel chromatography, to thereby to obtain a chemicalyield and an optical purity.

TABLE 12 (Synthesis by Continuous Recycling of Perfume (R-1-Octen-3-Ol)(Synthesis by Continuous Recycling of Perfume (R-1-Octen-3-Ol)Continuous Ketone R- S- Chemical Optical recycling oxide Alcohol Alcoholyield purity First time: 1.43 72.11 26.35 after 30 hours After 60 hours3.79 96.21 0 88.3% ≧98.13% ee Second time: 1.79 74.88 23.38 after 30hours After 60 hours 3.77 96.23 0 86.5% ≧98.10% ee Third time: 1.3877.33 21.29 after 30 hours After 60 hours 3.67 96.33 0 85.8% ≧98.22% eeFourth time: 1.92 77.1 20.98 after 30 hours After 60 hours 3.76 96.24 086.7% ≧98.28% ee Numerical value = GC % ratio Numerical value = GC %ratio

As is apparent from the above results, with respect to continuoussynthesis of substrate racemic 1-octen-3-ol, recycling can be carriedout at least 4 times and, since the yield of the formed ketone is lessthan 4% in any case and also the chemical yield is 85% or more, thederacemization reaction occurs. The optical purity was obtained with ahigh optical purity of 98% ee or more four times, and FIG. 5 shows theresults of GC/FID spectrum of R-1-octen-3-ol.

As is apparent from the above results, as shown in the followingreaction scheme, with respect to an aryl-based substrate including abenzene or naphthalene skeleton, 50% of the formed ketone is scarcelyreduced. With respect to an alkyl-chain substrate such as 1-octen-3-ol,there occurs deracemization wherein the formed ketone is furtherasymmetrically reduced.

Example 13 Determination of Molecular Weight of Protein Complex(SDS-Page)

FIG. 3 is SDS-page of a precipitate obtained by shaking a green pea-Caalginic acid gel for 10 hours or more in warm water and then allowingthe eluted protein complex to undergo crystallization precipitation atammonium sulfate 30%. An “enzyme solution” lane shown in the drawing isan aqueous warm solution itself eluted after shaking in warm water for10 hours or more, an “ammonium sulfate precipitate” lane is that whereina warm water solution is allowed to undergo crystallizationprecipitation so as to adjust to ammonium sulfate 30% saturated, and theprecipitate is redissolved by adding water, and a “supernatant” laneshows the results of SDS-page of the supernatant obtained by centrifugalseparation of the ammonium sulfate precipitate.

FIG. 4 shows an extract of “FIG. 5 (Influence of the addition of tableor common a salt on cross-linking efficiency by DST)” which has alreadybeen published in the description by Reiko Urade (Role of Table orCommon Salt in Network Formation of Gluten Protein) of “Food andTechnology”, 2008 December, General remarks. The drawing shows theresults obtained by ultrafugation of a solution, which is prepared bysolubilizing a dough made by adding a common or table salt to a wheatflour or not using an SDS solution containing β-mercaptoethanol, andcarrying out SDS-page with respect to a soluble fraction (S) and aninsoluble fraction (P). The insoluble fraction is treated with sodiummetaperiodate, separated by SDS polyacrylamide gel electrophoresis andthus stained with a protein.

With respect to Fig. A, since the molecular weight of a band arrangementof a fragment of an S—S bonded protein component cut by SDS of FIG. 3agrees with SDS-page shown in FIG. 3, it could be determined that theprotein complex is not composed of an enzyme, but is composed of apolymer, a low molecular glutenin, and a component similar to gliadin.Glutenin forms a disulfide bond between high-molecular and low-moleculargluten molecules by a function of air oxidation (and/or an exogenousenzyme) and thus undergoes polymerization. It is estimated that thisthiol-dithiol site becomes an active domain of a redox reaction and thuscauses asymmetric oxidation of a substrate racemic alcohol. It isconsidered that dissolved calcium not only induces formation of anintermolecular disulfide bond, but also is involved in aggregationbetween high-molecular and low molecular glutenin molecules (6.4 Å orless), and a property change to water solubility.

Example 14

FIG. 5 shows qualitative analytical results of samples (i) to (vi) usingFourier transform infrared spectrophotometry (FT-IR). Analysis wascarried out for the purpose of comparing a difference in molecularstructure between the respective samples. Sample information of samples(i) to (vi) is as follows.

(1) Green pea protein powder: obtained by the method described in theabove-mentioned Example 1(2) Sodium alginate powder: obtained from a commercially availableproduct(3) Cross-linked crystallized protein complex powder produced by shakingincubator: obtained by the method described in the below-mentionedExample 15(4) Cross-linked crystallized protein complex powder (iv) produced byjar fermentor: obtained by the method described in the below-mentionedExample 16(5) 20 mM Ca chloride/50 mM Tris HCl buffer (pH 6.0) and (pH 8.0):obtained by the method described in Example 17

As is apparent from the results shown in FIG. 5, a difference in Fouriertransform infrared spectrophotometry (FT-IR) between “samples (iii)(iv)” having an activity which catalyzes an asymmetric oxidationreaction and “samples (v) (vi)” which do not cause asymmetric oxidationreaction lie in two points, i.e. (1) absorption of a hydroxyl group(—O—Ca) of carboxylate (R—C(═O)—O—Ca) existing at 1,411 cm⁻¹, and (2)absorption of sugar ether (—C—O—C—) or ammonium sulfate (—O—S(═O)₂—O—)at 1082 cm⁻¹. Since strong absorption is observed in these two pointsfor “samples (iii) (iv)” having an activity which catalyze an asymmetricoxidation reaction, it is possible to explain assumed that (1)carboxylate derived from Ca alginate (R—C(═O)—O—Ca) and sugar ether(—C—O—C—) are incorporated into samples by encapsulating, or (2) sulfuroxide was formed in case of intermolecular disulfide bonding (—S—S—). Itis also considered that these two points become an important pointwhether or not “they have an activity which catalyzes the asymmetricoxidation reaction” and significance of encapsulating a crude protein inCa alginate exists.

Furthermore, FIG. 6 shows qualitative analytical results of samples (i)to (iv) using an X-ray microanalyzer EPMA-1600. Sample information ofsamples (i) to (iv) has already been explained.

As is apparent from the results of an X-ray microanalyzer of FIG. 6,sodium (Na) is detected in “sample (i)” having an activity whichcatalyzes an asymmetric oxidation reaction, but sodium (Na) is notdetected in “samples (ii) (iii)” having no activity which catalyzes anasymmetric oxidation reaction. Therefore, it is also estimated to bewreckage/footprint of Na alginate molecules of carboxylate(R—C(═O)—O—Na) wherein sodium (Na) derived from a green pea proteinpowder (iv) remains without being influenced by an action, or a sodium(Na) atom was not substituted with calcium (Ca) in the second step ofadding dropwise sodium alginate in a 4% calcium chloride solution, tothereby encapsulate in calcium alginate. Since the amount of sulfur (S)of the samples (i) to (iv) is remarkably large in case of (i), there isdisclosed a possibility that sulfur oxide (—O—S(═O)—O—) obtained throughformation of an intermolecular disulfide bond relatively causes anasymmetric oxidation reaction.

Therefore, it was possible to estimate that absorption of oxygen (O₂)described in the below-mentioned Example 19 was involved in formation ofan intermolecular disulfide bond (R1-S—S—R2) between cysteines (R1-S—HH—S—R2) in a green pea protein. When a sodium (Na) atom of sample (iv)is wreckage/footprint of the Na alginate molecule, there was suggested apossibility that (1) carboxylate derived from Ca alginate (R—C(═O)—O—Ca)and sugar ether (—C—O—C—) are incorporated in samples by encapsulating.

Example 15 Process for Producing Protein Complex Produced by ShakingIncubator Described in FIG. 5(iii) and FIG. 6(i)

In the second step, seeds were crushed and water was added and, after 1hour, a bean curd lee fraction of a precipitate was removed and awater-soluble fraction was spray-dried to obtain a protein powderderived from green pea (PP). To 30 g of the obtained protein powder, 300ml of distilled water corresponding to 10 times the equivalent weightwas added. After sufficiently dissolving under stirring, 300 ml of anaqueous 3% sodium alginate solution corresponding to 1 time theequivalent weight was added, followed by stirring until uniform. Theobtained green pea-sodium alginate mixed solution was added dropwise inan aqueous 4.0% calcium chloride solution (pH 9.16), to thereby obtaingel beads. Distilled water (500 ml) corresponding to 20 times theequivalent weight of the green pea protein powder as reaction solutionwas added to the obtained beads, followed by shaking at temperaturesetting of 40° C. and the number of shaking of 55 rpm of aconstant-temperature shaking incubator for 10 hours or more.

Apparatus Used

(1) Reaction tank: A 5,000 ml Erlenmeyer flask was respectively arrangedin a constant-temperature shaking incubator 3.

Number of shaking: 55 rpm

Temperature setting: 40° C.

Conditions of Stirring (Culturing) Operation

1. Green pea protein (30 g)-calcium alginate gel (about 600 mL)2. Distilled water: 500 mL×3 (compriseers)×3 (days)3. Number of extraction: 3 timee/3 days (recovered once a day)

Conditions of Extracting Operation

1. An aqueous culture solution for one day 500 mL×3 (compriseers) wasseparated and recovered from beads.2. Ammonium sulfate precipitate: An industrial ammonium sulfate is addedso as to adjust to 30% saturated ammonium sulfate, and then left tostand for crystallization precipitation for 1 day.3. Cross-linking: A 25% glutaraldehyde (GA) solution is added to acrystallized and precipitated solution so that the concentration becomes0.25%, and then left to stand for 2 hours or more.4. Centrifugal separation (washing): The precipitate is recovered at10,000 rpm over 15 minutes and the supernatant is removed, and thendistilled water is added, followed by washing impurities by repeatingtwice.5. Freeze-drying (powderization): carried out by a freeze dryer (FD).6. Asymmetric oxidation activity: A solution of racemic1-(2-naphthyl)ethanol (40 mL) having a concentration of 10 ppm preparedby dissolving 350 mg of a freeze-dried (FD) powder in 50 mM Tris-HClbuffer (pH 8.0) was added and oxidation activity was examined.7. Concentration of Ca (mg/g): After subjecting a powder sample after FDto an acid decomposition treatment, the amount of Ca was measured by anatomic absorption photometer (Contr. AA-300).

An example of a process for producing cross-linked crystallized proteincomplex using a constant-temperature shaking incubator is summarized inTable 12. Since a constant-temperature shaking incubator is equippedwith a 5,000 ml Erlenmeyer flask sealed simply with an aluminum foil,respectively, a culture solution is sufficiently shaken at the number ofshaking of 55 rpm and oxygen is sufficiently supplied. Polymerizationwas induced by a disulfide bond ((—S—S—)) through aglutenin-glutenin-cysteine residue (R1-S—H O H—S—R2) dehydrationcondensation reaction (→H₂O) and then carboxylate (R—C(═O)—O—Ca) derivedfrom Ca alginate and sugar ether (—C—O—C—) were incorporated byencapsulating. It is considered that an active domain formed in thisprotein complex allows R alcohol to undergo selective asymmetricoxidation in an enzyme-like manner, and thus making it possible toobtain S-alcohol having an optical purity of 100% e.e.

TABLE 13 (Activity Intensity of Protein Complex produced by ShakingIncubator described in FIG. 5(iii) and FIG. 6(i)) (Activity Intensity ofProtein Complex produced by Shaking Incubator described in FIG. 5(iii)and FIG. 6(i)) Three Total (once + twice + three Number of extractionOnce Twice times times): A Shaking time (hour) 24 24 24   72: AConcentration of Ca in 2.59 0.756 0.346 3.695: A extract solution(mg/mL) Different test of three times in total (A, B, C) A B C Yieldamount after FD — — — 6.4 20.3 20.6 (%) Concentration of Ca — — — 1.676.80 10.3 (mg/g) Asymmetric oxidation activity depending on reactiontime 7 hr 20 hr 7 hr 20 hr 7 hr 20 hr Oxidation of R-isomer — — — 3.3857.16 16.86 48.50 6.11 49.8 (%) Optical purity (% e.e.) 99.8 99.7 99.6Note) Concentration of Ca in extract solution (mg/mL): The concentrationof Ca of a shaken solution was measured by an atomic absorptionphotometer. Concentration of Ca (mg/g): The amount of Ca in a powderedsample after FD was measured by an atomic absorption photometer after anacid decomposition treatment. Oxidation of R-isomer (%): 7 hours and 20hours after reacting to a scale described in FIG. 9, the formed ketone(%) was determined by GC measurement and described. (Since 50% of anR-isomer of a substrate racemic alcohol is selectively oxidized intoketone, when ketone % is close to or more than 50%, an optical purity ofthe other S-alcohol is close to 100%) Optical purity (% e.e.):determined from “S-alcohol (area %) − R-alcohol (area %)” of GC.

Example 16 Process for Producing Cross-Linked Crystallized ProteinComplex Produced by 5 L Jar Fermentor described in FIG. 5(iv)

In the second step, seeds were crushed and water was added and, after 1hour, a bean curd lee fraction of the precipitate was removed and awater-soluble fraction was spray-dried to obtain a protein powderderived from green pea protein (PP). To 50 g of the obtained proteinpowder, 500 ml of distilled water corresponding to 10 times theequivalent weight was added. After sufficiently dissolving understirring, 500 ml of an aqueous 3% sodium alginate solution correspondingto 1 times the equivalent weight was added, followed by stirring untiluniform. The obtained green pea-sodium alginate mixed solution (1,000mL) was added dropwise in an aqueous 4.0% calcium chloride solution (pH9.16), to thereby obtain gel beads. To the obtained beads, distilledwater (1,000 mL) corresponding to 20 times the equivalent weight of thegreen pea protein powder was added as the reaction solution, followed byshaking at a temperature setting of 40° C. and number of shaking of 800rpm of a 5 L jar fermentor manufactured by Takasaki Kagaku Co., Ltd.

Apparatus Used

(1) Reaction tank: 5 L jar fermentor manufactured by Takasaki KagakuCo., Ltd.

Blade: two simple type bar-shaped paddler 800 rpm

Temperature: 40° C.

Oxygen supply: 0.5 mg/L

Conditions of Stirring (Culturing) Operation

4. Green pea protein (30 g)-calcium alginate gel (about 1,000 mL)5. Distilled water: 1, 000 mL×3 (days)6. Number of extraction: 3 times/3 days (recovered once a day)Conditions of Extracting Operation: An aqueous culture solution for oneday (1,000 mL) was separated and recovered from beads.1. Ammonium sulfate precipitate: An industrial ammonium sulfate is addedso as to adjust to 30% saturated ammonium sulfate, and then left tostand for crystallization precipitation for 1 day.2. Cross-linking: A 25% glutaraldehyde (GA) solution is added to acrystallized and precipitated solution so that the concentration becomes0.25%, and then left to stand for 2 hours or more.3. Centrifugal separation (washing): The precipitate is recovered at10,000 rpm over 15 minutes and the supernatant is removed, and thendistilled water is added, followed by washing impurities by repeatingtwice.4. Freeze-drying (powderization): carried out by a freeze dryer (FD).5. Asymmetric oxidation activity: A solution ofracemic-1-(2-naphthyl)ethanol (40 mL) having a concentration of 10 ppmprepared by dissolving 350 mg of a freeze-dried (FD) powder in 50 mMTris-HCl buffer (pH 8.0) was added and oxidation activity was examined.6. Concentration of Ca (mg/g): After subjecting a powder sample after FDto an acid decomposition treatment, the amount of Ca was measured by anatomic absorption photometer (Contr. AA-300).

As a result, as shown in Table 13, also in case of a 5 L jar fermentor,similar to the case of using a constant-temperature shaking incubator,it was considered that carboxylate (R—C(═O)—O—Ca) derived from theobjective Ca alginate and sugar ether (—C—O—C—) were incorporated byencapsulating, to thereby form an active domain in a green pea protein,and a protein complex allows R alcohol to undergo selective asymmetricoxidation in an enzyme-like manner, and thus making it possible toobtain S-alcohol having an optical purity of 100% e.e.

TABLE 14 (Activity Intensity of Protein Complex by 5L Jar Fermentordescribed in FIG. 5(iv)) (Activity Intensity of Protein Complex by 5LJar Fermentor described in FIG. 5(iv)) Three Number of extraction OnceTwice times Total Shaking time (hour) 24 24 24 72 Concentration of Ca in2.61 0.735 0.480 3.825 extract solution (mg/mL) Test A Crude yield 6.256.81 12.9 25.96 Yield amount after FD (%) — — — 10.4% (5.174 g)Concentration of Ca (mg/g) — — — 5.59 Asymmetric oxidation activitydepending on reaction 7 hr 20 hr time Oxidation of R-isomer (%) — — —17.57 47.99 Optical purity (% e.e.) 99.8 Note) Concentration of Ca inextract solution (mg/mL): The concentration of Ca of a shaken solutionwas measured by an atomic absorption photometer. Concentration of Ca(mg/g): The amount of Ca in a powdered sample after FD was measured byan atomic absorption photometer after an acid decomposition treatment.Oxidation of R-isomer (%): 7 hours and 20 hours after reacting to ascale described in FIG. 9, the formed ketone (%) was determined by GCmeasurement and described. (Since 50% of an R-isomer of a substrateracemic alcohol is selectively oxidized into ketone, when ketone % isclose to or more than 50%, an optical purity of the other S-alcohol isclose to 100%) Optical purity (% e.e.): determined from “S-alcohol (area%) - R-alcohol (area %)” of GC.

Example 17 Process for Producing “Cross-Linked Crystallized Protein (pH6.0 or pH 8.0)” Described in FIGS. 5(v) and 5(vi) and FIGS. 6(ii) and6(iii)

For the purpose of confirming necessity of the second step ofencapsulating in a calcium alginate gel, the second step (ofencapsulating in a calcium alginate gel) was omitted and a green peacrude protein (4 g) was allowed to undergo constant-temperature shaking(55 rpm, 40° C., 24 hours) using a 2% calcium chloride/50 mM Tris HClbuffer solution/120 ml ((v) pH 6.0 or (vi) pH 8.0). After shaking,ammonium sulfate was added to a green pea protein calcium solution so asto adjust to 0% saturated and, after being left to stand for 24 hours ormore (third step), the crystallized precipitate was cross-linked with0.25% glutaraldehyde (fourth step) and the obtained cross-linkedcrystallized green pea crude protein was freeze-dried (FD) to form apowder.

(12) Reaction tank: A 300 ml Erlenmeyer flask was respectively arrangedin a constant-temperature shaking incubator.

Number of shaking: 55 rpm

Temperature setting: 40° C.

Conditions of Stirring (Culturing) Operation

7. Green pea protein (4 g)8. 2% calcium chloride/50 mM Tris HCl buffer (120 m)9. Number of extraction: once/day

Conditions of Extracting Operation

1. Ammonium sulfate precipitate: Ammonium sulfate is added so as toadjust to 30% saturated ammonium sulfate after constant-temperatureshaking (55 rpm, 40° C., 24 hours).2. Cross-linking: A 25% glutaraldehyde (GA) solution is added to acrystallized and precipitated solution so that the concentration becomes0.25%, and then left to stand for 2 hours or more.3. Centrifugal separation (washing): The precipitate is recovered at10,000 rpm over 15 minutes and the supernatant is removed, and thendistilled water is added, followed by washing impurities by repeatingtwice.4. Freeze-drying (powderization): carried out by a freeze dryer (FD).5. Asymmetric oxidation activity: A solution ofracemic-(2-naphthyl)ethanol (40 mL) having a concentration of 10 ppmprepared by dissolving 350 mg of a freeze-dried (FD) powder in 50 mMTris-HCl buffer (pH 8.0) was added and oxidation activity was examined.6. Concentration of Ca (mg/g): After subjecting a powder sample after FDto an acid decomposition treatment, the amount of Ca was measured by anatomic absorption photometer (Contr. AA-300).

As shown in Table 15, it could be confirmed that the second step of“encapsulating in a calcium alginate gel” is indispensable for formationof a protein complex, in addition to the concentration of dissolved Caand dissolved oxygen which induce a glutenin-glutenin disulfide bond((—S—S—)).

TABLE 15 (Activity Intensity of “Protein Complex (pH 6.0 or pH 8.0)”described in FIGS. 5(v) and 5(vi) and FIGS. 6(ii) and 6(iii)) (ActivityIntensity of “Protein Complex (pH 6.0 or pH 8.0)” described in FIGS.5(v) and 5(vi) and FIGS. 6(ii) and 6(iii)) Sample at pH 6.0 Sample at pH8.0 Shaking time (hour) 24 24 Crude yield 12.3 11.8 Yield amount afterFD (%) 3.1 2.9 Concentration of Ca (mg/g) 15.1 20.6 7 hr 20 hr 7 hr 20hr Oxidation of R-isomer (%) 3.38 7.79 7.68 7.44 Optical purity (% e.e.)C C Note) Concentration of Ca (mg/mL): The amount of Ca in a powderedsample after FD was measured by an atomic absorption photometer.Oxidation of R-isomer (%): After 7 hours and 20 hours, the formed ketone(%) was determined by GC measurement and described. Asymmetric oxidationActivity C: Only an R-isomer of a racemic 1-(2-naphthyl)ethanol (10 ppm,40 ml) solution is scarcely asymmetrically oxidized by a “proteincomplex” (350 mg) within 20 hours (the formed ketone is 10% or less). B:Asymmetrically oxidation of only an R-isomer of a racemic1-(2-naphthyl)ethanol (10 ppm, 40 ml) solution is stopped by a “proteincomplex” (350 mg) during asymmetric oxidation until 20 hours (the formedketone is 30% or less). A: Only an R-isomer of a racemic1-(2-naphthyl)ethanol (10 ppm, 40 ml) solution is scarcelyasymmetrically oxidized by a “protein complex” (350 mg) within 20 hours(the formed ketone is 40% or more).

Example 18 Influence of Asymmetric Oxidation Activity (Right Axis) after6 Hours on Air Oxidation Time of Calcium Alginate Encapsulated Beads andon Protein Complex Yield in Second Step

FIG. 7 is a graph wherein the yield and oxidation activity of a proteincomplex are summarized; the protein complex being obtained by producingspherical green pea protein-calcium alginate beads are in accordancewith a usual method; leaving the beads left to stand in air for 0 hour,0.5 hour, 1 hour, 3 hours, 5 hours and 7 hours; carrying out shakingextraction in warm water through constant-temperature shaking incubation(40° C., 55 rpm) using a usual process for 2 days (first time) and 1 day(second time)) in the second step; leaving a warm water extract solutionto stand at saturated 30% ammonium sulfate for a whole day and night inaccordance with a usual method; cross-linking at the concentration of0.25% glutaraldehyde; freeze-drying the obtained cross-linkedcrystallized protein complex (CLDPs); and subjected cross-linkedcrystallized protein complex to ball mill crushing. With respect to theasymmetric oxidation activity, 10 ppm racemic 1-(2-naphthyl)ethanolsolution/50 mM Tris-HCl buffer (pH 8.0, 40 mL) was added to 350 mg ofthe cross-linked crystallized protein complex and oxidation activity isexhibited at 6 hours after constant-temperature shaking incubation (40°C., 55 rpm).

As is apparent from the above results, asymmetric oxidation activity ofthe cross-linked crystallized protein complex became strong as the airoxidation time becomes longer. In contrast, the yield of the obtainedprotein complex was high after 1 hour and 3 hours. Therefore, it couldbe confirmed that the protein complex induces (i) an intermoleculardisulfide bond (—S—S—) in a protein, (ii) intermolecular aggregation inprotein (shortening of an intermolecular distance: <6.4 Å), (iii) achange of property into water solubility and the like, to thereby forman active domain (Thioredoxin fold: Cys-X-Y-Cys sequences) together witha calcium salt in gel beads in a crude protein derived from animal andplant as an inexpensive material under an oxygen atmosphere by aninfluence of air oxidation (and/or an exogenous enzyme), and thus aprotein complex having an activity which suitably catalyzes anasymmetric oxidation reaction.

Example 19 Transition of pH and DO (Dissolved Oxygen Concentration) ofProtein Complex in 100 L Jar Production in Second Step

FIG. 9 is a drawing wherein the pH of distilled water (20 L) to be addedwhen a cross-linked crystallized protein complex is produced in thesecond step, and DO (dissolved oxygen) are monitored; the cross-linkedcrystallized protein complex being obtained by crushing seeds; addingwater; removing a bean curd lee fraction of the precipitate after 1hour; spray-drying a water-soluble fraction, to thereby obtain a greenpea protein powder; adding 10 L of distilled water corresponding to 10times the equivalent weight to 1 kg of the obtained protein powder inaccordance with a usual method; mixing them with stirring; adding 10 Lof an aqueous 3% sodium alginate solution corresponding to 10 times theequivalent weight; stirring the solution until uniform; and addingdropwise the obtained green pea-sodium alginate mixed solution (about 20L) in an aqueous 4.0% calcium chloride solution using a gel preparationdevice, to thereby form spherical beads having a diameter of about 1 to2 mm.

Apparatus Used

(2) Reaction tank: 400φ×700H (TL-TL), vertical cylindrical tank

Blade: 4 inclined puddles 200φ 35 rpm

Temperature: 40° C.

(3) Crystallization tank

420φ×460H, vertical cylindrical tank

Blade: 6 blades turbine 120φ

A. Conditions of Stirring (Culturing) Operation

1. Green pea protein (1 kg)-calcium alginate gel (about 20 L)2. Distilled water:First time—stirring (20 L) for 3 days (no oxygen supply), followed bythird and fourth stepsSecond time—stirring (20 L) for 2 days (no oxygen supply), followed bythird and fourth stepsThird time—stirring (20 L) for 1 day (oxygen supply), followed by thirdand fourth stepsFourth time—stirring (20 L) for 1 day (oxygen supply), followed by thirdand fourth steps

B. Conditions of Operation of Extracting Protein Complex

1. An aqueous culture solution (20 L) after stirring was separated andrecovered from beads.2. Ammonium sulfate precipitate: An industrial ammonium sulfate is addedso as to adjust to 30% saturated ammonium sulfate, and then left tostand for 1 day for crystallization precipitation.3. Cross-linking: A 25% glutaraldehyde (GA) solution is added to acrystallized and precipitated solution so that the concentration becomes0.25%, and then left to stand for 2 hours or more.4. Centrifugal separation (washing): The precipitate is recovered at10,000 rpm over 15 minutes and the supernatant is removed, and thendistilled water is added, followed by washing impurities by repeatingtwice.5. Freeze-drying (powderization): carried out by a freeze dryer (FD).6. Asymmetric oxidation activity: which represents ketone (%) obtainedby oxidizing an R-isomer by adding a solution of racemic1-(2-naphthyl)ethanol (40 mL) having a concentration of 10 ppm preparedby dissolving 350 mg of a freeze-dried (FD) powder in 50 mM Tris-HClbuffer (pH 8.0).7. Concentration of Ca (mg/g): After subjecting a powder sample after FDto an acid decomposition treatment, the amount of Ca was measured by anatomic absorption photometer (Contr. AA-300).

As shown in FIG. 9, with respect to beads produced by adding dropwise inan aqueous 4.0% calcium chloride solution (pH 9.16), in case of stirringin distilled water without supplying oxygen, the pH transited within aweak acid range, e.g., 5.72 (first time) and 6.20 (second time) anddissolved oxygen (DO) became zero after 0.7 hour (first time) and 1 hour(second time). In case of stirring while supplying oxygen (controlled to0.5 mg/L), the pH turned from weak acid to the weak alkali side, e.g.7.20 (third time). The pH always transited to weak alkali at about pH7.45 (fourth time) and DO transits within a range from about 0 to 1 mg/Lin both third and fourth times.

As is apparent from the above results, high dissolved Ca ionconcentration exists in the first and second times with no oxygensupply. It was examined whether or not formation of an active domain ina state where the dissolved oxygen concentration is zero (oxygendeficiency), i.e. formation of an active domain by a disulfide bondbetween glutenin molecules and the effect of shortening anintermolecular distance. However, activity was not recognized.Subsequently, 20 L of water was replaced in the third to fourth timesand, when a given dissolved oxygen concentration was maintained bysupplying airs in the presence of a low dissolved Ca ion concentration,it was examined whether or not formation of an active domain in a statewhere the dissolved oxygen concentration is zero (oxygen deficiency),i.e. formation of an active domain by a disulfide bond between gluteninmolecules and the effect of shortening an intermolecular distance.However, while weak oxidation activity is also recognized in this case,the optical purity of the formed alcohol did not exceed 50% ee.Therefore, it could be confirmed that it becomes an important point tomaintain the dissolved Ca ion concentration and given dissolved oxygenconcentration in formation of an active domain formed by a disulfidebond between glutenin molecules and the effect of shortening anintermolecular distance.

(Influence of Asymmetric Oxidation Activity (Right Axis) after 6 Hourson Air Oxidation Time of 3% Calcium Alginate Encapsulated Beads and onProtein Complex Yield (Left Axis) in Second Step)

FIG. 7 is a graph wherein the yield and oxidation activity of a proteincomplex are summarized, the protein complex being obtained by producingspherical green pea protein-calcium alginate beads in accordance with ausual method; leaving the beads to stand in air for 0 hour, 0.5 hour, 1hour, 3 hours, 5 hours and 7 hours; carrying out shaking extraction inwarm water through constant-temperature shaking incubation (40° C., 55rpm) using a usual process for 2 days (first time) and 1 day (ninthtime); leaving a warm water extract solution to stand at saturated 30%ammonium sulfate for a whole day and night in accordance with a usualmethod; cross-linking at the concentration of 0.25% glutaraldehyde;freeze-drying the obtained cross-linked crystallized protein complex(CLDPs); and subjected the freeze-dried cross-linked crystallizedprotein complex to ball mill crushing. With respect to the asymmetricoxidation activity, 10 ppm racemic 1-(2-naphthyl)ethanol solution/50 mMTris-HCl buffer (pH 8.0, 40 mL) was added to 350 mg of the cross-linkedcrystallized protein complex and oxidation activity is exhibited at 6hours after constant-temperature shaking incubation (40° C., 55 rpm).

Example 18a Influence of Asymmetric Oxidation Activity (Right Axis)after 6 Hours on Air Oxidation Time of 3% Calcium Alginate EncapsulatedBeads and on Protein Complex Yield (Left Axis) in Second Step

FIG. 7 is a graph wherein the yield and oxidation activity of a proteincomplex are summarized; the protein complex being obtained by producingspherical green pea protein-calcium alginate beads are produced inaccordance with a usual method; leaving the beads to stand in air for 0hour, 0.5 hour, 1 hour, 3 hours, 5 hours and 7 hours; carrying outshaking extraction in warm water through constant-temperature shakingincubation (40° C., 55 rpm) using a usual process for 2 days (firsttime) and 1 day (ninth time); leaving a warm water extract solution tostand at saturated 30% ammonium sulfate for a whole day and night inaccordance with a usual method; cross-linking at the concentration of0.25% glutaraldehyde; freeze-drying the obtained cross-linkedcrystallized protein complex (CLDPs); and subjecting the freeze-driedcross-linked crystallized protein complex to ball mill crushing. Withrespect to the asymmetric oxidation activity, 10 ppm racemic1-(2-naphthyl)ethanol solution/50 mM Tris-HCl buffer (pH 8.0, 40 mL) wasadded to 350 mg of the cross-linked crystallized protein complex andoxidation activity is exhibited at 6 hours after constant-temperatureshaking incubation (40° C., 55 rpm).

As is apparent from the above results, asymmetric oxidation activity ofthe cross-linked crystallized protein complex became strong as the airoxidation time becomes longer. In contrast, the yield of the obtainedprotein complex constantly transited between about 11% to 15% when morethan 1 hour has passed, and the yield focused to 12% to 13%. Therefore,it could be confirmed that that the protein complex induces (i) anintermolecular disulfide bond (—S—S—) in a protein, (ii) intermolecularaggregation in protein (shortening of an intermolecular distance: <6.4Å), (iii) a change of property into water solubility and the like, tothereby form an active domain (Thioredoxin fold: Cys-X-Y-Cys sequences)together with a calcium salt in gel beads in a crude protein derivedfrom animal and plant as an inexpensive material under an oxygenatmosphere by an influence of air oxidation (and/or an exogenousenzyme), and thus a protein complex having an activity which suitablycatalyzes an asymmetric oxidation reaction.

Example 20 Influence of Asymmetric Oxidation Activity (Right Axis) after7 Hours on Na Alginate Concentration (0.5%, 1%, 1.5%, 2%, 3%) of CalciumAlginate Encapsulated Beads and on Protein Complex Yield in Second Step

FIG. 13 is a graph wherein the yield and oxidation activity of a proteincomplex are summarized; the protein complex being obtained by producingspherical green pea protein-calcium alginate beads (Na alginateconcentration (0.5%, 1%, 1.5%, 2%, 3%) in accordance with a usualmethod; leaving the beads to stand in air for 7 hours; carrying outshaking extraction in warm water through constant-temperature shakingincubation (40° C., 55 rpm) using a usual process for 2 days (firsttime) and 1 day (second time); leaving a warm water extract solution tostand at saturated 30% ammonium sulfate for a whole day and night inaccordance with a usual method; cross-linked at the concentration of0.25% glutaraldehyde; freeze-drying the obtained cross-linkedcrystallized protein complex (CLDPs); freeze-drying the cross-linkedcrystallized protein complex; and subjecting the freeze-driedcross-linked crystallized protein complex to ball mill crushing. Withrespect to the asymmetric oxidation activity, 10 ppm racemic1-(2-naphthyl)ethanol solution/50 mM Tris-HCl buffer (pH 8.0, 40 mL) wasadded to 350 mg of the cross-linked crystallized protein complex andoxidation activity is exhibited at 6 hours after constant-temperatureshaking incubation (40° C., 55 rpm).

As is apparent from the above results, the yield of the obtained proteincomplex is the highest when the Na alginate concentration is 1.5%. Evenif the concentration increases or decreases from the above value, theyield gradually decreased (0.5%<1%<1.5%>2%>3%). In contrast, oxidationactivity of the protein complex slightly increased (0.5%<1%<1.5%<2%<3%)as the Na alginate concentration increased. Therefore, it becameapparent that an improvement in yield can be expected by allowing aprotein-calcium alginate gel produced by setting the Na alginateconcentration at about 1.5% to undergo air oxidation for 5 to 7 hours ormore, to thereby obtain cross-linked crystallized protein complexes(CLPCs) in accordance with a usual method.

Example 21 Difference in Asymmetric Oxidation Rate of Protein Complex(50 mg, 75 mg, 100 mg, 200 mg, 300 mg) to Substrate Concentration (10ppm)

FIG. 14 shows the results wherein 40 ml of 50 mM Tris-HCl (pH 8.0)buffer of racemic 1-(2-naphthyl)ethanol having a concentration of 10 ppmis added to a freeze-dried (FD) powder (50 mg, 75 mg, 100 mg, 200 mg,300 mg) of the protein complex obtained in Example 20 in a 200 mlErlenmeyer flask and the reaction is carried out by shaking in warmwater using a constant-temperature shaking incubator (40° C., 55 rpm),and then a persistence ratio (%) of R-isomer-1-(2-naphthyl)ethanol to beasymmetrically oxidized is traced every 4 hours by GC.

As is apparent from the above results, the reaction rate increasedrelative to the asymmetric oxidation reaction as the addition amount ofthe protein complex as the catalyst increased more and more relative tothe substrate concentration (10 ppm) (50 mg>75 mg>100 mg>200 mg>300 mg).Since the protein complex reaction is a dehydrogenation reaction whichdoes not depend on a coenzyme NAD (P), unlike a conventionaldehydrogenation reaction which depends on baker's yeast, cultured plantcells, and coenzyme NAD (F) of microorganism cells. Therefore, it isconsidered that the catalyst is a catalyst wherein not only reduction inmaterial cost, but also reduction in reaction cost can be expected

Example 21a Difference in Asymmetric Oxidation Rate of SubstrateConcentration (10-30 ppm, 30-90 ppm, 90-110 ppm, 110-140 ppm) to ProteinComplex (300 mg)

FIG. 15 shows the results wherein 40 ml of 50 mM Tris-HCl (pH 8.0)buffer of racemic 1-(2-naphthyl)ethanol having a concentration (10-140ppm) is added to a freeze-dried (FD) powder (300 mg) of the proteincomplex obtained in Example 20 in a 200 ml Erlenmeyer flask and thereaction is carried out by shaking in warm water using aconstant-temperature shaking incubator (40° C., 55 rpm), and then apersistence ratio (%) of R-isomer-1-(2-naphthyl)ethanol to beasymmetrically oxidized is traced every 4 hours by GC.

As is apparent from the above results, the asymmetric oxidation reactionof the substrate concentration (10 to 140 ppm) to the protein complex(300 mg) of the catalyst was classified into four patters 10-30 ppm,30-90 ppm, 90-110 ppm and 110-140 ppm, as shown in FIG. 15. That is, itwas found that the reaction time of 20 hours is required in case of thesubstrate concentration of 10-30 ppm, 40 hours is required in case of30-90 ppm, 60 hours in case of 90-110 ppm, and 140 hours in case of110-140 ppm. Therefore, it was found that the reaction time is fast asthe concentration of the substrate becomes lower, and the reaction timebecomes drastically low at the concentration of 110 ppm or more.

INDUSTRIAL APPLICABILITY

It is possible to produce an optical isomer such as a optically activealcohol to be synthesized used in the fine chemical fields such aspharmaceuticals, perfumes and foods, at low cost in an environmentallyfriendly and easy manner, by using the first step of freeze-drying (FD)animal and plant resources, for example, grains such as buckwheat,amaranth, rice, wheat, barley, corn, oats, rye, foxtail millet, barnyardmillet, millet, adlay and sorghum; beans such as adsuki beans, kidneybeans, green peas, green beans and soy beans, and the respective planttissue of (husks (brans, rice brans), germs (sprouts), leaves (youngleaves, sprouts), stems, roots and flowers of grasses and weeds furtherincluded therein, and also the respective animal tissue of egg whitederived from animal, and muscle, to thereby finely crush, and optionallydissolving in warm water, removing husks, and spray-drying awater-soluble moiety, to thereby concentrate a water-soluble protein;the second step of encapsulating the crude protein in a calcium alginategel, to thereby allow the protein to undergo air oxidation,blank-shaking in warm water, and extracting the objective proteincomplex from the gel; the third step of crystallizing the proteincomplex using 30% saturated ammonium sulfate, to thereby form aprecipitate; the fourth step of cross-linking the precipitated proteincomplex; and the fifth step of freeze-drying (FD) the obtained proteincomplex, to thereby form a powder in combination.

1. A process for producing a cross-linked crystallized protein complex,comprising: a first step of concentrating a crude protein from awater-soluble moiety derived from an animal or plant; a second step ofencapsulating the protein in a gel, to thereby allow the protein toundergo air oxidation, and then extracting a protein complex from thegel; a third step of allowing the extracted protein complex to undergocrystallization and precipitation in an aqueous solution; and a fourthstep of cross-linking the precipitated protein complex.
 2. The methodaccording to claim 1, wherein the protein complex is a protein complexcontaining at least protein and calcium.
 3. The method according toclaim 1, wherein the protein complex further comprises a saccharide. 4.The method according to claim 1, wherein the protein complex is aprotein complex having an activity in catalyzing an asymmetric oxidationreaction.
 5. The process for producing a protein complex according toclaim 1, a tissue derived from animal or plant is freeze-dried (FD),finely crushed, and then a crude protein eluted with water isconcentrated by precipitation in the first step.
 6. The process forproducing a protein complex according to claim 1, wherein a tissuederived from animal or plant is freeze-dried (FD), finely crushed, andthen a crude protein eluted with water is concentrated by spray-dryingin the first step.
 7. The process for producing a protein complexaccording to claim 1, wherein the gel in the second step is a calciumalginate gel.
 8. The process for producing a protein complex accordingto claim 1, wherein air oxidation in the second step is carried out byleaving the gel to stand in air.
 9. The process for producing a proteincomplex according to claim 1, wherein a protein complex is extracted inwarm water from the gel in the second step.
 10. The process forproducing a protein complex according to claim 7, wherein theconcentration of an aqueous calcium chloride solution for preparing thecalcium alginate gel is 50 mM or more.
 11. The process for producing aprotein complex according to claim 7, wherein the amount of warm waterwhen the calcium alginate gel is shaken in warm water in the second stepis 1.5 times or less the amount of the calcium alginate gel.
 12. Theprocess for producing a protein complex according to claim 1, whereinair oxidation in the second step is involved in an oxidation activityintensity.
 13. The process for producing a protein complex according toclaim 1, wherein the gel is left to stand in air for 30 minutes or more,to thereby allow the gel to undergo air oxidation in the second step.14. The process for producing a protein complex according to claim 1,wherein the air oxidation exerts the effect of enhancing formation of aprotein complex and water solubility of the complex.
 15. The process forproducing a protein complex according to claim 11, wherein the step ofshaking in warm water is carried out using a constant-temperatureshaking incubator in the second step.
 16. The process for producing aprotein complex according to claim 11, wherein the second step, the stepof shaking in warm water is carried out using a jar fermentor.
 17. Theprocess for producing a protein complex according to claim 1, whereinthe protein complex is a protein complex having a property similar tothose of a high molecular glutenin, a low molecular glutenin and gliadinin the second step.
 18. The process for producing a protein complexaccording to claim 1, wherein the protein complex has a property ofaggregating in the presence of a trace amount of a salt, to therebyshorten an intermolecular distance in the second step.
 19. The processfor producing a protein complex according to claim 18, wherein a traceamount of the salt is a salt which gives calcium ions.
 20. The processfor producing a protein complex according to claim 1, wherein ammoniumsulfate is used when the protein complex obtained in the third step iscrystallized.
 21. The process for producing a protein complex accordingto claim 20, wherein the concentration of the ammonium sulfate issaturated 30% or less.
 22. The process for producing a protein complexaccording to claim 1, wherein the cross-linking agent which allows aprotein complex to undergo cross-linking crystallization in the fourthstep is glutaraldehyde (GA) and/or formaldehyde (FA).
 23. The processfor producing a cross-linked crystallized protein complex according toclaim 1, wherein the plant in the first step is selected from grainssuch as buckwheat, amaranth, rice, wheat, barley, corn, oats, rye,foxtail millet, barnyard millet, millet, adlay and sorghum; and beanssuch as adsuki beans, kidney beans, green peas, green beans and soybeans.
 24. The process for producing a protein complex according toclaim 23, wherein the grains and beans comprise a plant resource of seedhusks, germs, leaves, stems, roots and flowers obtained as a tissue atthe time of crushing.
 25. The process for producing a protein complexaccording to claim 1, wherein the animal in the first step is selectedfrom chickens, amphibians, fishes, egg albumin derived from chicken egg,and muscle.
 26. A process for producing an optically active alcohol,which comprise reacting a racemic alcohol as a substrate with a proteincomplex, to thereby selectively obtain one enantiomer of the racemicalcohol.
 27. A process for producing an optically active alcohol, whichcomprise asymmetrically oxidizing one enantiomer of a substrate racemicalcohol into ketone stereoselectively using a protein complex, tothereby obtain the other enantiomer, which is not involved in thereaction, with a high optical purity.
 28. A protein complex containingat least a protein, which has at least one peak in a region of (1,085±50cm⁻¹) and a region of (1,411±50 cm⁻¹), respectively, in FT-IR.
 29. Theprotein complex according to claim 28, which has an activity incatalyzing an asymmetric oxidation reaction.
 30. The protein complexaccording to claim 28, which further comprises a saccharide.
 31. Theprotein complex according to claim 28, wherein the protein is across-linked crystallized protein.
 32. The protein complex according toclaim 29, wherein the asymmetric oxidation reaction is an oxidationreaction which selectively gives one enantiomer of a substrate racemicalcohol.
 33. The protein complex according to claim 29, which has anactivity which gives a deracemization reaction which enables the ketoneobtained by the asymmetric oxidation reaction to be further reducedasymmetrically.